Detection of missense mutations by single-strand conformational polymorphism (SSCP) analysis in five dysfunctional variants of coagulation factor VII

Five unrelated subjects with dysfunctional coagulation factor VII (FVII) were studied In order to Identify missense mutations affecting function. Exons 2 to 8 and the Intron-exon Junctions of their FVIl genes were amplified from peripheral white blood cell DNA by PCR and screened by SSCP analysis. DNA fragments showing aberrant mobility were sequenced. The following mutations were Identified: In case 1 (FVII: C <1%, FVIl:Ag 18%) a heterozygous A to G transltion at nucleotlde 8915 In exon 6 results In the amlno acid substitution Lys-137 to Glu near the C-termlnus of the FVlla llght chaln; In case 2 (FVII: C 7%, FVll:Ag 47%) a heterozygous A to G transltion at nucleotide 7834 In exon 5 results in the substitution of Gin-100 by Arg in the second EGF-like domain; In case 3 (FVll:C 20%, FVIl:Ag 76%) a homozygous G to A transition at nucleotide position 6055 in exon 4 was detected resulting in substitution of Arg-79 by Gin in the first EGF-like domain; in case 5 (FVIl:C 10%, FVIl:Ag 52%) a heterozygous C to T transition at nucleotide position 6054 in exon 4 also results in the substitution of Arg79, but in this case it is replaced by Trp; case 4 (FVll:C <1%, FVIl:Ag 100%) was homozygous for a previously reported mutation (G to A) at nucleotide position 10715 in exon 8, substituting Gin for Arg at position 304 in the protease domain. Cases 1,2 and 5 evidently have additional undetected mutation

Adenovirus-Associated Virus Vector-Mediated Gene Transfer in Hemophilia B

NIHR (RP-PG-0310-1001), the Medical Research Council, the Katharine Dormandy Trust, the U.K. Department of Health, NHS Blood and Transplant, the NIHR Biomedical Research Centers (to University College London Hospital and University College London), the ASSISI Foundation of Memphis, the American Lebanese Syrian Associated Charities, the Howard Hughes Medical Institute, the National Heart, Lung, and Blood Institute (HL094396), the Royal Free Hospital Charity Special Trustees Fund 35, the Royal Free Hospital NHS Trust, and St. Jude Children’s Research Hospita

α1-antitrypsin Pittsburgh in a family with bleeding tendency

We describe a 16-year-old girl and her 41-year-old father who both had a bleeding tendency, dramatic prolongation of all standard clotting assays, undetectable levels of plasma protein C activity, and low or borderline levels of factors X, XI and XII. Plasma and serum electrophoresis revealed a minor peak following the main α1 globulin peak, of which the proportion was increased. Platelet aggregation by thrombin (final concentration 1 U/mL) was absent in both patients, but this inhibition can be overcome by increasing the concentration of thrombin (4 U/mL). The molecular defect responsible for these coagulation abnormalities was identified by genomic sequencing. Both patients are heterozygous for α1-antitrypsin Met 358 to Arg (α1-antitrypsin Pittsburgh). Seven other members of this pedigree had normal coagulation tests and do not carry the same genetic mutation. This unique family with α1-antitrypsin Pittsburgh sheds some light on the study of this extremely rare mutation and its inheritance

Human tissue factor pathway inhibitor fused to CD4 binds both FXa and TF/FVIIa at the cell surface

Tissue factor pathway inhibitor (TFPI) is one of the main regulators of the tissue factor (TF) pathway of coagulation. To tether human TFPI to the cell surface, full length or truncated TFPI lacking the third Kunitz domain were fused with domains three and four and the carboxy-terminal sequence of human CD4. Constructs were transfected into a mouse fibroblast cell line and individual clones were checked for expression using monoclonal antibodies directed against the first two TFPI Kunitz domains and against CD4. Specific human FXa binding was detected by flow cytometry using an anti-FX polyclonal antibody, and inhibition of FXa proteolytic activity was verified by chromogenic substrate assay using S-2765. In addition, TFPI-CD4-expressing cells, preincubated with FXa, specifically bound human TF-FVIIa complexes as revealed with an anti-human TF polyclonal antibody. No functional difference was observed between full length or truncated TFPI-CD4. These results demonstrate that functionally intact TFPI can be tethered to the cell surface. Genetic manipulation of, for example, endothelial cells leading to the stable expression of TFPI may inhibit the development of coronary artery heart disease following cardiac allotransplantation, and may inhibit thrombosis in the context of xenotransplantation

Mathematical models of coagulation—are we there yet?

BackgroundMathematical models of coagulation have been developed to mirror thrombin generation in plasma, with the aim of investigating how variation in coagulation factor levels regulates hemostasis. However, current models vary in the reactions they capture and the reaction rates used, and their validation is restricted by a lack of large coherent datasets, resulting in questioning of their utility.ObjectivesTo address this debate, we systematically assessed current models against a large dataset, using plasma coagulation factor levels from 348 individuals with normal hemostasis to identify the causes of these variations.MethodsWe compared model predictions with measured thrombin generation, quantifying and comparing the ability of each model to predict thrombin generation, the contributions of the individual reactions, and their dependence on reaction rates.ResultsWe found that no current model predicted the hemostatic response across the whole cohort and all produced thrombin generation curves that did not resemble those obtained experimentally. Our analysis has identified the key reactions that lead to differential model predictions, where experimental uncertainty leads to variability in predictions, and we determined reactions that have a high influence on measured thrombin generation, such as the contribution of factor XI.ConclusionThis systematic assessment of models of coagulation, using large dataset inputs, points to ways in which these models can be improved. A model that accurately reflects the effects of the multiple subtle variations in an individual’s hemostatic profile could be used for assessing antithrombotics or as a tool for precision medicine

University–Industry Linkages in Egypt: A Political Economy Approach

Submitted in partial fulfilment of the requirements for the degrees Master in Economic Analysis and Policy (APE), University of Paris 13 and University of Paris 7 – Diderot Master of Commerce in Development Theory and Policy, University of the Witwatersrand Under the Erasmus Mundus Joint Master Degrees Programme Economic Policies in the Age of Globalisation (EPOG)The Egyptian Science, Technology and Innovation (STI) sector has witnessed several institutional changes in the last ten years. It is slowly becoming more oriented toward satisfying the industrial Research and Development (R&D) needs with a more competitive approach to R&D expenditure. Meanwhile, the state has been in political turbulence since 2011 with a subsequent economic/financial crisis. In this thesis, we ask how the institutional changes that seek to stimulate Technology Transfer (TT) from the R&D institutions to the industrial sector can be understood within a broader political economy context. We break down our question into four main investigations. The first is concerning the economic rents and governance mechanisms as dictated by the state’s industrial policy. The second investigates the relationship between the state and the industrialists given the distribution of power among economic factions while considering changes, if any, in the Political Settlements (PS) before and after 2011. The third question describes the innovation patterns in the manufacturing sector and the financial and knowledge flows within the National System of Innovation (NSI). Finally, we present a case study for the TT Offices (TTOs), providing a narration of their institutional weaknesses. For our analysis, we use a hybrid framework by merging the PS and the NSI approaches. The former is used to check for the compatibility of the industrial policy in Egypt with the prevailing distribution of power among the state factions while the second provides a framework for analyzing financial and knowledge linkages in the Egyptian NSI as well as the University-Industry Linkages (UILs). The research methodology adopted in this thesis is mixed and involves literature review and qualitative analysis of data from conducted interviews. The methodology first relies on analyzing the available literature on the industrial policies and political economy in Egypt. The investigation then proceeds to give a descriptive account of the Egyptian NSI from which we move forward to present the case for TT from university to industry by conducting five semi-structured interviews with officials from TTOs belonging to R&D institutions. Results of our investigation reveal that the variation in manufacturing sector performance is linked with the distribution of economic rents by the state to the capitalists, in return of political support provided by the latter to the former. Hence, in line with the predictions of the PS theory, the industrial policy failed to realize higher competitiveness of the Egyptian manufacturing sector in the international market as the supporting industrial faction (capitalists) had accumulated enough power to weaken the state’s capacity of implementing its active industrial policy. We note that the variation in sector performance reflects not only growth but also innovation rates. While sectors receiving higher economic rents show less rates of innovation, except for beverage industry, those with less rents show the highest 3 innovation rates. The core contribution of the thesis comes with presenting the case for TTOs in Egypt which were recently established to induce UILs. Conducted interviews reveal that the STI policy is still in an early phase of formulation and that the organizational capacity of the STI actors remains low and constricted by various institutional and legal barriers. We infer, from other country experiences, that the PS in Egypt, which has persisted for a long time and without significant changes after 2011, does not indicate substantial changes in the power distribution among its state factions and hence, in the incentives structure and governance mechanisms towards a successful implementation of an industrial policy. Hence, we conclude that while the STI institutions are being reformed slowly, the prevailing PS and the associated inclination towards neoliberal economic policies in which the STI sector is being shaped in Egypt do not allow for an efficient TT through the free-market mechanisms, as perceived by the state.XL201

Expression of hirudin fusion proteins in mammalian cells : A strategy for prevention of intravascular thrombosis

Background - Intravascular thrombosis occurs in disorders of diverse pathogeneses, including allograft and xenograft rejection. In this in vitro study, we describe an approach for tethering the specific thrombin inhibitor hirudin to plasma membranes as part of a genetic strategy for regulating intravascular coagulation. Methods and Results - An HLA class I leader sequence was fused with hirudin linked to domains 3 and 4 of human CD4 and intracytoplasmic sequence from either CD4 or human P-selectin. The constructs were transfected into mouse fibroblasts, Chinese hamster ovary (CHO)-K1 cells, immortalized porcine endothelial cells (IPECs), and a pituitary secretory cell line (D16/16). Thrombin binding to the hirudin fusion proteins expressed on fibroblasts and CHO-K1 cells could be blocked by an anti- hirudin monoclonal antibody and by pretreatment of thrombin with either the synthetic tripeptide thrombin inhibitor PPACK or native hirudin. Hirudin expression significantly modified the procoagulant phenotype of IPECs in human plasma, leading to prolongation of clotting times. Hirudin-CD4-P- selectin fusion proteins accumulated in adrenocorticotropic hormone- containing granules in D16/16 cells, with no cell surface expression except on activation with phorbol ester, when hirudin relocated to the outer membrane. Conclusions - Hirudin fusion proteins were expressed on mammalian cells, where they reduced local thrombin levels and inhibited fibrin formation. Regulated expression was achieved on activated cells by use of the cytoplasmic sequence from P-selectin. In vivo, these fusion proteins may prove useful transgenic or gene therapy agents for preventing intravascular thrombosis