Thesis (PhD)--University of South Australia, 2000

Thesis (M.Med.Sc.) -- University of Adelaide, Dept. of Clinical and Experimental Pharmacology, 2000. / Bibliography: leaves 115-125.

Thesis (MTech(Medical Technology))--Cape Technikon, 1997 / Cryoprecipitates are used as the raw material for the preparation of Factor VIII (FVIIIl) for replacement therapy for haemophiliacs. Routinely, cryoprecipitate only recovers 50% of the Factor VIII in the plasma. The purpose of this study, production of cryoprecipitate, was to investigate those variables which play a key role in determining the yield of Factor VIII present in cryoprecipitate. Cryoprecipitate production involves a wide range of variables which could effect the final outcome of the product. These vary from the donor blood group, time of donation, exercise levels of the donor, to a time delay prior to processing, temperature storage conditions, to the method utilised for plasma freezing and thawing. The objective was to explore which combination of variables in the procedure would lead to a process which would optimize the preparation of cryoprecipitate in a routine environment, to yield the highest levels of Factor VIII. Frequently in scientific investigations, particularly when a practical approach has to be adopted, questions arise in which the effects of a number of different variables in a process, require evaluation. Such questions can usually be most economically investigated, by arranging the analysis according to an ordered plan in which all the factors are viewed in a regular way. Provided the plan has been correctly chosen, it is possible to determine not only the effect of each individual variable, but also the way in which each effect depends on the other factor, by means of an interaction. This makes it possible to obtain a more complete picture of what is happening, than would have been obtained by varying each of the variables one at a time while keeping the others constant. Designs of this sort lend themselves well to statistical analysis, and provide their own estimates of experimental error. This type of statistical analysis called, 2K Fractional Factorial Experimental Design, forms the basis of this study in which 14 key variables in the production process of cryoprecipitate were defined as possible areas in which Factor VIII levels in the cryoprecipitate are effected. Key variables have been identified on an individual basis in previous studies (Burka et al., 1975), however this blended approach to optimise the key variables within the production environment, and define further combinations which could be incorporated into the production, has never been attempted. The statistical design used in the study was compiled by the Institute for Biostatistics of the Medical Research Council (MRC). Units of blood were collected and processed, from blood donors under the stipulated criteria, corresponding to the study design.

Factor VIII, a component of the intrinsic pathway of blood coagulation, has yet to be purified to homogeneity. It appears that, in vivo, the factor VIII coagulant protein is closely associated with one or more other proteins (factor VHI-related antigen and platelet aggregating factor). The material normally isolated from bovine plasma as 'factor VIII' possesses all three activities and is therefore either a mixture or a complex of the various proteins. In the present study, bovine factor VIII:C was purified approximately fivethousand- fold by a combination of ion-exchange chromatography and fractional precipitation. The factor VIII coagulant activity can be separated from the other activities of the 'factor VIII complex' but the procedures involved are not suitable for preparative use as the factor VIII:C which is obtained is unstable. During coagulation, factor VIII:C is required during the activation of factor X. Studies with purified bovine clotting factors indicate that factor IX_a is the enzyme responsible for the cleavage of factor X, in a calcium-dependent reaction which is stimulated by phospholipid. Factor VIII:C further accelerates the rate at which factor X_a is generated. Preliminary investigations of the kinetic parameters of the reaction indicate that the stimulation by factor VIII:C occurs through a marked increase in the V_max of the reaction; factor VIII:C does not affect the K_m for factor X. The coagulant activity of factor VIII is enhanced by exposure to thrombin, but the 'activated' factor VIII:C which is produced is not itself capable of activating factor X in the absence of factor IX_a. Thus, the 'activation' of factor VIII:C, in contrast to the activation of, for example, factors IX and X, does not appear to result in the formation of an enzyme. That is, factor VIII:C is a non-enzymic, high molecular weight cofactor for factor IX_a.

The role of the nonionic surfactant Tween 80 on the behavior of the therapeutic recombinant protein Factor VIII (rFVIII) was investigated at solid/liquid and air/water interfaces. In order to provide a model system to compare results obtained for the complicated rFVIII-Tween system, a well-characterized globular protein lysozyme was used. The experimental scheme involved the introduction of the protein and Tween to the adsorption substrate in different manners, either lysozyme Tween together or in sequence as lysozyme followed by Tween or vice versa. It was observed that the addition of Tween together with lysozyme reduced the amounts adsorbed at hydrophobic surfaces, while no such reduction was observed on hydrophilic surfaces. A high Tween concentration was required to effect the removal of the lysozyme molecules from the hydrophobic surface and Tween was not effective in removing lysozyme from the hydrophilic surface at any concentration. These results suggest that the Tween-surface interaction is important in determining lysozyme adsorption. Similar observations were made for the rFVIII-Tween system at hydrophobic and hydrophilic silica interfaces. In this case, the presence of interfacial and solution Tween together resulted in complete prevention of rFVIII adsorption. Electrostatic forces were observed to be play an important role in rFVIII adsorption. The rFVIII-Tween interactions at solid interfaces were also evaluated using intrinsic fluorescence and biological activity measurements. Results obtained with respect rFVIII adsorbed mass, and structure or biological activity change upon adsorption, were evaluated in parallel. This parallel evaluation suggested that rFVIII adsorption on hydrophilic, negatively charged surfaces is likely to be highly ordered and oriented in a manner that retains the solvent accessibility of the active sites in rFVIII. On the other hand, rFVIII may adsorb to hydrophobic surfaces in different orientations, with a likelihood of surface induced unfolding. rFVIII-Tween interaction at the air/water interface was investigated separately. Surface tension data recorded for rFVIII-Tween mixtures suggested that Tween dominated the air/water interface as the Tween concentration was increased. Reduced interface-induced unfolding was observed at high Tween concentrations. These results were also thought to contribute to the reduction in rFVIII aggregation typically observed as a result of exposure to the air/water interface. / Graduation date: 2006

Thesis (M.S.)--Oregon State University, 2010. / Printout. Includes bibliographical references (leaves 42-44). Also available on the World Wide Web.

Factor VIII is a clotting factor found on the intrinsic side of the coagulation cascade. A mutation in the factor VIII gene causes the disease Hemophilia A, for which there is no cure. The most common treatment is administration of recombinant factor VIII. However, this can cause an immune response that renders the treatment ineffective in certain hemophilia patients. For this reason a new treatment, or cure, needs to be developed. Gene editing is one solution to correcting the factor VIII mutation. CRISPR/Cas9 mediated gene editing introduces a double stranded break in the genomic DNA. Where this break occurs repair mechanisms cause insertions and deletions, or if a template oligonucleotide can be provided point mutations could be introduced or corrected. However, to accomplish this goal for editing factor VIII mutations, a way to deliver the components of CRISPR/Cas9 into somatic cells is needed. In this study, I confirmed that the CRISPR/Cas9 system was able to create a mutation in the factor VIII gene in zebrafish. I also showed that the components of CRISPR/Cas9 could be piggybacked by vivo morpholino into a variety of blood cells. This study also confirmed that the vivo morpholino did not interfere with the gRNA binding to the DNA, or Cas9 protein inducing the double stranded break.