PREFACTOR-IX - THE PROPEPTIDE INHIBITS BINDING TO MEMBRANE SURFACES AND ACTIVATION BY FACTOR-XI

The gamma-carboxylase recognition site in the propeptide of prefactor IX signals the gamma-carboxylation of specific glutamic acid residues in the adjacent Gla domain during factor IX biosynthesis. To study posttranslational processing of the vitamin K-dependent blood coagulation factors and the properties of processing intermediates, we have isolated an incompletely processed factor IX species, prefactor IX, from the medium of heterologous mammalian cells expressing the human factor IX cDNA. Prefactor IX was purified by sequential immunoaffinity chromatography using antibodies specific for the propeptide and antibodies specific for the well-carboxylated factor IX species. This purified prefactor IX preparation was fully gamma-carboxylated and contained the N-terminal propeptide, but it exhibited no factor IX procoagulant activity. Prefactor IX was not cleaved following incubation with factor Xla. In contrast to mature factor IX, prefactor IX did not demonstrate Ca(II)-dependent binding to acidic phospholipid vesicles, nor can the membrane binding surface be expressed, as detected by antibodies specific for this epitope. The propeptide of prefactor IX can be removed in vitro by a specific endopeptidase, furin/PACE, yielding factor IX, which can be converted to fully active factor IXa by factor XIa and which binds normally to acidic phospholipid vesicles. These results indicate that fully gamma-carboxylated prefactor IX is biologically inactive due to the presence of the propeptide.