Recombinant human factor X: High yield expression and the role of furin inproteolytic maturation in vivo and in vitro

Factor X/Xa plays a pivotal role in the coagulation cascade and exhibits a therapeutic potential for the treatment of factor X-deficient as well as FV III and FIX inhibitor patients. This report describes the establishment of Chinese hamster ovary cell clones expressing recombinant human factor X up to 120 mu g/mL x day and 78 mu g/10(6) cells x day, that is to 100-fold hig her levels than reported previously. Although propeptide removal and single chain precursor to light and heavy chain processing as well as vitamin K-d ependent gamma-carboxylation became impaired at these expression levels, up to 25%, of the recombinant human factor X produced was active. This repres ents the highest functional activity ever reported for a vitamin K-dependen t protein at such an expression level. Expression of recombinant human fact or X in Chinese hamster ovary cells lacking the endoprotease Furin revealed that propeptide removal still occurred, whereas single chain precursor to light/heavy chain processing was abolished. This suggests that a protease d ifferent from Furin mediates propeptide removal, a unique finding compared with the other vitamin K-dependent coagulation factors. In contrast, exposu re of incompletely processed rFX molecules to soluble recombinant Furin in vitro mediated both of these cleavage reactions despite the absence of a ty pical arg(P4)-x(P3)-lys/arg(P2)-arg(P1) Furin cleavage site in the propepti de, indicating relaxed specificity in vitro. Concomitantly with the degree of processing, the functional activity of recombinant human factor x increa sed. Interestingly, Furin was shown to even perform correct N-terminal prot eolytic trimming of FX molecules truncated amino-terminal to the P3 residue in vitro. Depending on the absence or presence of warfarin in the culture media, as well as on the processing state, four distinct recombinant human factor x light chain isoforms were observed and their structure characteriz ed. One of these light chain farms correlated with the functional activity. Finally, the distribution of the individual light chain isoforms suggests that gamma-carboxylation may be a prerequisite for propeptide removal. (C) 2000 Elsevier Science Ltd. All rights reserved.