Effect of von Willebrand factor coexpression on the synthesis and secretion of factor VIII in Chinese hamster ovary cells.

Abstract

In plasma, antihemophilic factor (factor VIII) exists as a 200-kilodalton heavy-chain polypeptide in a metal ion association with an 80-kilodalton light-chain polypeptide. This complex is bound by hydrophobic and hydrophilic interactions to a large multimeric glycoprotein, von Willebrand factor (vWF). Accumulation of secreted human factor VIII activity expressed in Chinese hamster ovary cells requires the addition of serum in the growth medium, which provides vWF. Here we report that coexpression of vWF with factor VIII in Chinese hamster ovary cells resulted in increased stable accumulation of factor VIII activity in the absence of serum in the growth medium. In the coexpressing cells, the vWF cDNA transcription unit was transcribed to yield mRNA which was efficiently translated. vWF was properly processed and secreted to yield disulfide-bonded high-molecular-weight multimers similar to those observed in vWF secreted from human endothelial cells. Nuclear run-on assays showed that the factor VIII gene was transcribed at a level similar to that of the vWF gene, but the mRNA did not accumulate to high levels in the cytoplasm. In addition, although the translation efficiency of the factor VIII mRNA was similar to that of vWF, the processing and secretion of the factor VIII primary translation product was dramatically reduced compared with vWF. These results demonstrate that in Chinese hamster ovary cells both factor VIII mRNA accumulation and the processing and secretion of the primary factor VIII translation product are inefficient processes.The factor VIII complex has two distinct biologic functions: coagulant activity and a role in primary hemostasis (for reviews, see references 32 and 43). The analysis of factor VIII deficiency diseases, classic hemophilia and von Willebrand's disease, have contributed to the understanding that factor VIII is a complex of two components: the factor VIII procoagulant protein (antihemophilic factor) and the factor VIII-related antigen (von Willebrand factor [vWF]).The factor VIII molecule is an important regulatory protein in the blood coagulation cascade. After activation by thrombin, it accelerates the rate of factor X activation by factor IXa, eventually leading to the formation of the fibrin clot (18,28,39,49,50). Factor VIII is synthesized as a large precursor which is cleaved to generate an amino-terminalderived heavy chain of 200 kilodaltons (kDa) in a metal ion-stabilized complex with the carboxy-terminal-derived light chain of 80 kDa (9,13,41,51). Factor VIII has binding sites for factor IXa, factor X, Ca2 , phospholipid, and vWF.

Expression of Factor VIII by Murine Liver Sinusoidal Endothelial Cells

“…This represents the first demonstration of homologous expression of fVIII mRNA in primary cell culture. In contrast, previous studies of fVIII expression in cell culture have been conducted by transfecting fVIII gene fragments into liver cellderived cell lines (21,22) or Chinese hamster ovary cells (23,24). Our results should facilitate studies of fVIII gene regulation under more physiological conditions.…”

Analysis of factor VIII mediated suppression of lentiviral vector titres

“…The development of gene transfer systems for treating haemophilia A have been hindered by the low expression of FVIII which is two orders of magnitude lower than for other proteins. 1 This is ascribed to the presence of transcriptional silencers within the FVIII cDNA [2][3][4] and inefficient intracellular transport and secretion of the protein. 5 The construction of retroviral vectors encoding FVIII has been especially problematic and is attributed to inefficient transcription of the vector genome.…”

Gene therapy for hemophilia

“…Using a comparable retroviral vector delivery system, the levels of F.VIII produced in vitro were about 2 orders of magnitude lower than F.IX (218). The inefficiency of F.VIII production results from several factors, including inefficient expression of the mRNA, misfolding and degradation of the translated protein, and retention in the endoplasmic reticulum via binding to ER chaperones such as immunoglobulin binding protein (BiP) (218)(219)(220)(221)(222)(223)(224)(225).…”

“…Studies in vitro have suggested that this stabilizing effect is enhanced when F.VIII and vWF are co-expressed in the same cell, rather than simply adding vWF to the media (219,228). However, while vWF is believed to be produced in endothelial cells, platelets, and megakaryocytes, the site of F.VIII synthesis is somewhat controversial (229)(230)(231).…”