Revised Manuscript Received August 4, 1994® abstract: The y-carboxylase recogmtion site in the propeptide of profactor IX signals the y-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 factorsand the properties of processing intermediates, we have isolated an incompletely processed factor IX species, profactor IX, from the medium of heterologous mammalian cells expressing the human factor IX cDNA. Profactor IX was purified bysequential immunoaffinity chromatography using antibodies specific for the propeptide and antibodies specific for the well-carboxylated factor IX species. This purified profactor IX preparation was fully y-carboxylated and contained the N-terminal propeptide, but it exhibited no factor IX procoagulant activity. Profactor IX was not cleaved following incubation with factor XIa. In contrast to mature factor IX, profactor 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 profactor 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 inactive due to the presence of the propeptide.