FIRST EPIDERMAL GROWTH FACTOR-LIKE DOMAIN OF HUMAN BLOOD-COAGULATION FACTOR-IX IS REQUIRED FOR ITS ACTIVATION BY FACTOR VIIA TISSUE FACTOR BUT NOT BY FACTOR XIA

Factor IX consists of a gamma-carboxyglutamic acid-rich domain followe d by two epidermal growth factor (EGF)-like domains and the C-terminal protease domain. To delineate the function of EGF1 domain in factor I X, we constructed three mutants: an EGF1 domain-deleted mutant (IX(Del ta EGF1)), a point mutant (IX(Q50P)) with a Gln-50 --> Pro change, and a replacement mutant (IX(PCEGF1)) in which the EGF1 domain of factor IX was replaced by that of protein C. These mutants and wild-type (WT) factor IX (IX(WT)) were expressed in 293 kidney cells by using pRc/CM V vector. The purified proteins had the same gamma-carboxyglutamic aci d content as the normal plasma factor IX (IX(NP)) and were activated n ormally by factor XIa-Ca2+ Tn contrast, IX(Delta EGF1) could not be ac tivated by factor VIIa-tissue factor-Ca2+, and the activation of IX(PC EGF1) in this system was markedly slow; however, IX(Q50P) was activate d at a normal rate. In additional studies, both IX(WT) and IX(Delta EG F1) were rapidly converted to their respective IX alpha forms by facto r Xa-phospholipid-Ca2+. Since this reaction has an absolute requiremen t for phospholipid, it indicates that the mutants under study are not impaired in their interactions with phospholipid. Relative coagulant a ctivities of factor XIa-activated proteins were IX(NP), 100%; IX(WT), 75-85%; IX(Delta EGF1), less than or equal to 1%; IX(PCEGF1), less tha n or equal to 2%; and IX(Q50P), 6-10%. We conclude that the EGF1 domai n of factor IX is required for its activation by factor VIIa-tissue fa ctor and that the Gln-50 residue is not critical for this activation. Further, the EGF1 domain of factor IX is not essential for phospholipi d binding and for its activation by factor XIa. In addition, the low c oagulant activities of the activated mutants indicate that the EGF1 do main is also important in factor X activation by factor IXa-factor VII Ia-Ca2+-phospholipid complex.