SummaryHemophilia 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 procedure resulted in 5,300-fold enrichment and a 70% yield. Highly purified recombinant murine fVIII had a specific coagulant activity of 660 units per nanomole. It underwent proteolytic processing by thrombin to yield an activated heterotrimer that demonstrated significantly greater stability than activated human fVIII. Recombinant murine fVIII was utilized to generate an anti-fVIII polyclonal antibody. Intravenous injection of recombinant murine fVIII into hemophilia A mice failed to induce a significant anti-fVIII immune response using a schedule that yielded high titer inhibitory antibodies to human fVIII. This may provide an important model for the study of immune tolerance to fVIII.

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 R484, R489, and P492. The immunogenicity of purified recombinant B domaindeleted (BDD) human factor VIII molecules containing mutations at R484A/ R489A or R484A/R489A/P492A was studied in hemophilia A mice. Inhibitory antibody titers in mice receiving the R484A/R489A/P492A mutant, but not the R484A/R489A mutant, were significantly lower than in mice receiving control human BDD factor VIII. The specific coagulant activity and the in vivo clearance and hemostatic efficacy in hemophilia A mice of the R484A/R489A/P492A mutant were indistinguishable from human BDD factor VIII. Thus, the inhibitory antibody response to human factor VIII can be reduced by mutagenesis of a B-cell epitope without apparent loss of function, suggesting that this approach may be useful for developing a safer form of factor VIII in patients with hemophilia A. (Blood. 2004; 104:704-710)

To cite this article: Parker ET, Craddock HN, Barrow RT, Lollar P. Comparative immunogenicity of recombinant B domain-deleted porcine factor VIII and Hyate:C in hemophilia A mice presensitized to human factor VIII. J Thromb Haemost 2004; 2: 605-11.Summary. Hyate is a commercial plasma-derived porcine factor (F)VIII concentrate that is used in the treatment of patients with inhibitory antibodies to FVIII. OBI-1 is a recombinant B domain-deleted form of porcine FVIII that is in clinical development for the same indication. Hemophilia A mice were presensitized with human FVIII to simulate clinical inhibitory antibody formation and then were randomized to receive OBI-1 or Hyate:C in a comparative immunogenicity trial. OBI-1 or Hyate:C were given in a series of four intravenous injections at weekly intervals at doses of 1, 10, or 100 U kg . Inhibitory antibodies to porcine FVIII were not detected by Bethesda assay in most of the mice given OBI-1 or Hyate:C at doses of 1 or 10 U kg )1 , but were identified in 81% and 94% of mice given 100 U kg )1 of OBI-1 or Hyate:C, respectively. There was no significant difference between OBI-1 and Hyate:C in inhibitory antibody formation at any dose, although there was a trend toward a lower Bethesda titer in OBI-1-treated mice at 10 U kg )1 (P ¼ 0.09). Total anti-FVIII antibodies to Hyate:C and OBI-1 were also measured by ELISA using immobilized purified plasma-derived porcine FVIII and OBI-1, respectively, as antigens. At the 10 and 100 U kg )1 doses, the mean anti-FVIII response was higher in Hyate:C-treated-mice than in OBI-1-treated mice (P ¼ 0.02 and P ¼ 0.004, respectively). The results using this model suggest that OBI-1 may be less immunogenic and safer than Hyate:C in FVIII inhibitor patients.

Fulminant hepatic failure (FHF) in humans produces a bleeding diathesis due in large part to a reduction in the biosynthesis of liver-derived coagulation factors. Remarkably, factor VIII procoagulant activity is elevated in most of these patients despite widespread liver cell death. FHF can be modeled in mice by administration of azoxymethane, the active ingredient found in cycad palm nuts. We compared the expression of factor VIII to other hepatic hemostatic factors in azoxymethane-induced murine FHF. Mice displayed dose-dependent decreases in all coagulation factor activities measured, including factors V, VII, VIII, and IX. At the highest dose of azoxymethane (50 g/g body weight), factor VIII activity in plasma decreased by 98% within 36 hours after treatment, which was associated with an 80% reduction in hepatic factor VIII messenger RNA (mRNA). In contrast, factor VIII mRNA levels in spleen, kidney, and lung tissue of azoxymethane-treated mice were unchanged. Cellular damage in these mice appeared to be limited to hepatocytes as evident by histologic examination. This finding is supported by 2 observations. First, hepatic mRNA levels of von Willebrand factor, which is synthe- IntroductionDespite more than 50 years of investigation, the nature of factor VIII biosynthesis in vivo remains poorly understood. [1][2][3][4] Factor VIII messenger RNA (mRNA) is present in nearly every tissue examined, including liver, spleen, lymph node, heart, brain, lung, kidney, testes, muscle, and placenta. [5][6][7] The liver clearly is an important site of synthesis because liver transplantation cures human and canine hemophilia A. [8][9][10] However, the relative contribution of hepatocytes and liver sinusoidal endothelial cells remains uncertain. 7,[11][12][13][14][15] Furthermore, the contribution of extrahepatic biosynthesis of factor VIII in health and disease states is not known.Paradoxically, there usually is a significant rise in plasma factor VIII activity in human fulminant hepatic failure (FHF). [16][17][18][19] This increase can be more than 14 times normal levels and occurs simultaneously with profound decreases in the circulating levels of vitamin K-dependent and fibrinolytic plasma proteins. 16,20,21 The analysis of this phenomenon has been hindered by the lack of a reproducible animal model of FHF. However, a murine model of FHF recently was developed using the hepatotoxin azoxymethane (AOM), which is found in cycad palm nuts on the island of Guam. 22 AOM-treated mice develop hepatocellular necrosis, elevated liver transaminase and ammonia levels in blood and hepatic encephalopathy. They die within days, with death related to the dose of AOM. In this study, we examined the expression of factor VIII and several other hemostatic factors, including factors V, VII, and IX, and von Willebrand factor (VWF) in AOM-induced murine FHF. Materials and methods MaterialsMale C57BL/6 mice (22-30 g body weight) were purchased from Charles River Laboratory (Wilmington, MA). AOM and TriReagent were purchased from Sigma (St Lou...