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Patients with hemophilia A present with spontaneous and sometimes life-threatening bleeding episodes that are treated using blood coagulation factor VIII (fVIII) replacement products. Although effective, these products have limited availability worldwide due to supply limitations and product costs, which stem largely from manufacturing complexity. Current(More)
Clinical success for gene therapy of hemophilia A will be judged by achievement of sustained, therapeutic levels of coagulation factor VIII (fVIII). Previous clinical trials have suffered from transient, subtherapeutic expression of human fVIII transgenes. Porcine fVIII contains sequence elements that enable more efficient biosynthesis than human fVIII due(More)
Recombinant human factor VIII expression levels, in vitro and in vivo, are significantly lower than levels obtained for other recombinant coagulation proteins. Here we describe the generation, high level expression and characterization of a recombinant B-domain-deleted porcine factor VIII molecule. Recombinant B-domain-deleted porcine factor VIII expression(More)
Approximately 25% of patients with hemophilia A develop inhibitory antibodies after treatment with factor VIII. Most of the inhibitory activity is directed against epitopes in the A2 and C2 domains. Anti-A2 inhibitory antibodies recognize a 25-residue segment bounded by R484-I508. Several antigenic residues in this segment have been identified, including(More)
The stability of activated human and porcine factor VIII (fVIII) differ, but a direct comparison of their structural and functional properties has not been made. Highly purified, heterodimeric human recombinant and porcine plasma-derived fVIII were exchanged into a common buffer and some minor contaminants were removed by anion-exchange chromatography. The(More)
Hemophilia A is the inherited bleeding disorder that results from mutation of blood coagulation factor VIII (fVIII). Described here is the generation of a regulated expression system producing recombinant murine fVIII. Murine B-domainless fVIII was expressed at a peak level of 4 units/106 cells/24 h in serum-free media. Subsequently, a two-step purification(More)
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(More)
Blood coagulation factor VIII has a domain structure designated A1-A2-B-ap-A3-C1-C2. Human factor VIII is present at low concentration in normal plasma and, comparably, is produced at low levels in vitro and in vivo using transgenic expression techniques. Heterologous expression of B domain-deleted porcine factor VIII in mammalian cell culture is(More)
Human and porcine factor VIII (fVIII) are activated by thrombin to form a heterotrimer composed of subunits designated A1 and A2 derived from the fVIII heavy chain (HC) and a subunit designated A3-C1-C2 derived from the fVIII light chain (LC). Human and porcine fVIII were activated at the same rate to the same peak levels but dissociation of the A2 subunit(More)
We developed a quantitative method to study the efficacy of intravenously delivered human factor VIII in hemophilia A mice. Mortality was assessed after tail transection under conditions in which there were no survivors in untreated hemophilia A mice. Blood loss was significantly greater in untreated hemophilia A mice compared to normal C57BL/6 mice, and in(More)