The diversity of factor VIII (fVIII) C2 domain antibody epitopes was investigated by competition enzyme-linked immunosorbent assay (ELISA) using a panel of 56 antibodies. The overlap patterns produced 5 groups of monoclonal antibodies (MAbs), designated A, AB, B, BC, and C, and yielded a set of 18 distinct epitopes. Group-specific loss of antigenicity was associated with mutations at the Met2199/ Phe2200 phospholipid binding ␤-hairpin (group AB MAbs) and at Lys2227 (group BC MAbs), which allowed orientation of the epitope structure as a continuum that covers one face of the C2 ␤-sandwich.

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-domaindeleted porcine factor VIII expression levels are 10-to 14-fold greater than recombinant B-domain-deleted human factor VIII levels by transient and stable expression in multiple cell lines. Peak expression of 140 units⅐10 6 cells ؊1 ⅐24 h ؊1 was observed from a baby hamster kidneyderived cell line stably expressing recombinant porcine factor VIII. Factor VIII expression was performed in serum-free culture medium and in the absence of exogenous von Willebrand factor, thus greatly simplifying protein purification. Real time reverse transcription-PCR analysis demonstrated that the differences in protein production were not caused by differences in steady-state factor VIII mRNA levels. The identification of sequence(s) in porcine factor VIII responsible for high level expression may lead to a better understanding of the mechanisms that limit factor VIII expression.Factor VIII (fVIII) 1 is a large (ϳ 300 kDa) glycoprotein that functions as an integral component of the intrinsic pathway of blood coagulation. Mutations in the fVIII gene that result in decreased or defective fVIII protein give rise to the genetic disease, hemophilia A, which is phenotypically characterized by recurrent bleeding episodes. Treatment of hemophilia A entails intravenous infusion of either human plasma-derived or recombinant fVIII material. Approximately 25% of all hemophilia A patients treated with fVIII products develop antibodies that inhibit fVIII activity and limit treatment efficacy (1). Patients with fVIII-inhibitory antibodies can be treated using porcine plasma-derived fVIII products, which generally display low cross-reactivity with the human fVIII antibodies (2, 3). Currently there is not a recombinant porcine fVIII product available for clinical use.Since the introduction of recombinant fVIII for the treatment of hemophilia A, commercial suppliers have struggled to keep up with high patient demand (4). The shortage of recombinant fVIII material has precluded prophylactic treatment of severely affected patients, limited the implementation of immune-tolerance regimens, and kept treatment costs high. Unfortunately, fVIII is expressed and recovered at low levels in the heterologous mammalian cell culture systems used for commercial manufacture. The importance of this problem has fueled significant research efforts to overcome the low fVIII expression barrier, and several basic mechanisms have been identified that limit fVIII expression (for review, see Kaufman et al. (5)) Despite these findings, fVIII expression levels remain low, and a product shortage persists.The porcine fVIII cDNA sequence has been reported and shown to encode the homology-defined internal protein domain structure, A1-A2-B-ap-A3-C1-C2 (6, 7). Porcin...

Autosomal dominant polycystic kidney disease (ADPKD) largely results from mutations in the PKD1 gene leading to hyperproliferation of renal tubular epithelial cells and consequent cyst formation. Rodent models of PKD suggest that the multifunctional hormone insulin-like growth factor-1 (IGF-1) could play a pathogenic role in renal cyst formation. In order to test this possibility, conditionally immortalized renal epithelial cells were prepared from normal individuals and from ADPKD patients with known germline mutations in PKD1. All patient cell lines had a decreased or absence of polycystin-1 but not polycystin-2. These cells had an increased sensitivity to IGF-1 and to cyclic AMP, which required phosphatidylinositol-3 (PI3)-kinase and the mitogen-activated protein kinase, extracellular signal-regulated protein kinase (ERK) for enhanced growth. Inhibition of Ras or Raf abolished the stimulated cell proliferation. Our results suggest that haploinsufficiency of polycystin-1 lowers the activation threshold of the Ras/Raf signalling system leading to growth factor-induced hyperproliferation. Inhibition of Ras or Raf activity may be a therapeutic option for decreasing tubular cell proliferation in ADPKD.