Thomas Dock Steenstrup

Recombinant coagulation factor VIIa – from molecular to clinical aspects of a versatile haemostatic agent

Recombinant factor VIIa (rFVIIa, NovoSeven) is currently the only bypassing agent produced by recombinant technology for the treatment of haemophiliacs whose disease is complicated by inhibitory antibodies. In addition, recombinant production of FVIIa has made it widely available for a variety of purposes and accelerated the growth of our knowledge about FVIIa by generating an abundance of clinical and biochemical data. This fascinating molecule has turned out to be a safe haemostatic agent with great potential in the clinic and has inspired the generation of improved variants currently in (pre-)clinical testing. The present review describes the structural and functional aspects of FVIIa, followed by sections dealing with the manufacturing, therapeutic mechanism of action, clinical development and experience with rFVIIa.

All post-translational modifications except propeptide cleavage are required for optimal secretion of coagulation factor VII

Human coagulation factor VII (FVII) has two N-glycosylation sites (N145 and N322) and two O-glycosylation sites (S52 and S60). In transiently transfected COS-7 cells, all combinations of N- and O-glycosylation knock-out mutations reduced the release of FVII to the medium. Pulse-chase analysis of CHO-K1 cell lines expressing recombinant FVII demonstrated that virtually all wild-type FVII synthesized was secreted from the cells, whereas both N- and O-glycosylation knock-out mutations induced partial intracellular degradation of the synthesized FVII. Likewise, two thirds of the FVII synthesized in vitamin K-depleted and warfarin-treated CHO cells was degraded intracellularly, demonstrating the importance of gamma-carboxylation for the secretion of FVII. The furin inhibitor decanoyl-R-V-K-R-chloromethylketone inhibited propeptide cleavage, but FVII with propeptide appeared to be secreted equally well as FVII without propeptide. Propeptide cleavage was not inhibited by vitamin K depletion and warfarin treatment, suggesting that for FVII, correct gamma-carboxylation is not required for optimal processing of the propeptide. In conclusion, all post-translational modifications of FVII except propeptide cleavage were important for complete secretion of the synthesized FVII and to avoid intracellular degradation. Thus, the extensive post-translational modification of FVII seems critical for the intracellular stability of the protein and is required for keeping the protein in the secretory pathway.

More than one intracellular processing bottleneck delays the secretion of coagulation factor VII

Coagulation factor VII (FVII) is a vitamin K-dependent glycoprotein that undergoes extensive post-translational modification prior to secretion. Secretion of FVII proteins from producer cells is a slow process. To identify bottlenecks for the transport of FVII through the secretory pathway of FVII-producing cells, we analysed the processing of intracellular FVII by pulse-chase of FVII producing CHO cells followed by radioimmuno precipitation, SDS-PAGE, and autoradiography. FVII was coprecipitated with GRP78 and vice versa for at least three hours after synthesis of the labelled FVII, suggesting that nascent FVII is retained in the endoplasmic reticulum (ER). Judged from barium citrate precipitation assay, gamma-carboxylation of the pulse-labelled FVII was a slow process requiring several hours and seemed to be the most important bottleneck in the intracellular processing of FVII. Nevertheless, FVII was not released from the cells immediately after gamma-carboxylation. Gamma-carboxylated FVII accumulated in the cells and migrated as a band with reduced mobility compared to uncarboxylated FVII. This shift in migration was caused by N-glycan processing in the Golgi complex. Thus, the release of FVII from producer cells is delayed by at least two bottlenecks. The major bottleneck appears to be gamma-carboxylation, which determines the rate of transport of FVII out of the ER. Another bottleneck retains FVII in the cells after processing of the N-glycans into complex chains. Cells with an intact gamma-carboxylation machinery appear to posses mechanisms that protect nascent FVII from intracellular degradation and keep FVII in the ER until it is gamma-carboxylated.