Biological activity of recombinant factor VIII variants lacking the central B-domain and the heavy-chain sequence Lys713-Arg740: discordant in vitro and in vivo activity.

Recombinant factor VIII variants with overlapping deletions spanning the region Lys713-Ile1668 have been expressed in mammalian cells, and analysed for biological activity both in vitro and in vivo. Two distinct assay systems were used to measure the activity in vitro. The one-stage coagulation assay served to assess factor VIII procoagulant activity while a spectrophotometric assay was used for the quantification of factor VIII cofactor activity in factor IXa-dependent factor X activation. Deletion of the entire B-domain (Ser741-Arg1648) resulted in a protein with similar procoagulant and cofactor activity. In contrast, factor VIII-del(713-1637), which has a deletion that also comprises the heavy-chain sequence Lys713-Arg740, had lost factor VIII procoagulant activity while factor VIII cofactor activity was retained. This functional inconsistency was further addressed by comparing purified factor VIII-del(713-1637) with factor VIII-del(868-1562), a mutant with normal in vitro activity. Kinetic studies of factor Xa formation revealed that higher concentrations of thrombin were required to develop the cofactor activity from factor VIII-del(713-1637) than needed for factor VIII-del(868-1562) or plasma factor VIII. The physiological significance of this finding was assessed in dogs with haemophilia A. Both deletion mutants were similar to plasma factor VIII with regard to binding to von Willebrand factor and half-life and recovery. Employing the cuticle bleeding time model, factor VIII-del(868-1562) was found to be indistinguishable from plasma factor VIII, whereas factor VIII-del(713-1637) was less effective. The increased thrombin-resistance of factor VIII-del(713-1637) thus limits both procoagulant activity and haemostatic efficacy in cuticle bleeding. These observations suggest that the heavy-chain sequence Lys713-Arg740, although dispensable for factor VIII cofactor function per se, is involved in the proteolytic activation of factor VIII both in vitro and in vivo.

The uptake and expression of the factor VIII and reporter genes by vascular cells.

The conditions and efficacy of transfection of vascular cells in primary culture using DEAE-dextran, calcium phosphate and lipofectin have been investigated using chloramphenicol acetyltransferase and luciferase as reporter genes. Subsequently factor VIII was expressed in endothelial and smooth muscle cells. Both reporter genes could be expressed after transfection of umbilical vein endothelial cells, umbilical artery smooth muscle cells and fibroblasts. The expression of both reporter genes in endothelial and smooth muscle cells was highest using lipofectin. After transfection of smooth muscle cells with both full-length and mutant factor VIII genes, factor VIII activity and antigen were secreted into the culture medium, the secretion remaining stable to serial cell passage. The secretion of factor VIII from transfected smooth muscle cells was confirmed by the immunoprecipitation of [35S]methionine labelled protein. Endothelial cells also were successfully transfected with the mutant factor VIII gene.