BACULOVIRUS-MEDIATED EXPRESSION OF THE EPIDERMAL GROWTH FACTOR-LIKE MODULES OF HUMAN FACTOR-IX FUSED TO THE FACTOR-XIIIA TRANSAMIDATION SITE IN FIBRONECTIN - EVIDENCE FOR A DIRECT INTERACTION BETWEEN THE NH(2)-TERMINAL EPIDERMAL GROWTH FACTOR-LIKE MODULE OF FACTOR-IXA-BETA AND FACTOR-X

Factor IX is a vitamin K-dependent procoagulant zymogen of a serine pr otease. In the presence of Ca2+ the active form of factor IX (factor I Xabeta) forms a complex with factor VIIIa on suitable phospholipid sur faces such as aggregated platelets. This macromolecular complex rapidl y activates factor X. We have previously provided data that suggest an interaction between the NH2-terminal epidermal growth factor (EGF)-li ke module of factor IXabeta and the substrate factor X. In an alternat ive approach to study this protein-protein interaction, we have expres sed three recombinant baculovirus constructs encoding the EGF-like mod ules of human factor IX and a truncated form of fibronectin in a syste m based on the infection of insect cells (Spodoptera frugiperda 21). T his strategy allows a simple one-step purification of the recombinant proteins on a gelatin-Sepharose column, followed by removal of the gel atin-binding part derived from fibronectin by proteolytic cleavage. Th e fusion proteins were isolated at yields of 20-50 mug/ml culture medi um. The recombinant EGF-like modules contained 0.2-0.4 mol of erythro- beta-hydroxyaspartic acid/mol of protein, i.e. similar to the amount f ound in factor IX from human plasma, and appeared to be glycosylated a t Ser-53. The NH2-terminal EGF-like module, which contained a transami dation acceptor site derived from ribronectin, was cross-linked by fac tor Xllla in solution to intact and Gla-domainless factor X. There was no evidence of cross-linking to activated factor X or to factor X fra gments containing only the gamma-carboxyglutamic acid module and the t wo EGF-like modules. The cross-linking results suggest a specific inte raction between the NH2-terminal EGF-like module of factor IXabeta and the heavy chain of unactivated factor X. This interaction, albeit wea k as judged by competition experiments, may be important for the targe ting of factor X to the factor IXabeta-factor VIIIa complex on biologi cal membranes and for the subsequent dissociation of factor Xa from th e complex after activation.