FACTOR-IX ZUTPHEN - A CYS(18)-]ARG MUTATION RESULTS IN FORMATION OF AHETERODIMER WITH ALPHA(1)-MICROGLOBULIN AND THE INABILITY TO FORM A CALCIUM-INDUCED CONFORMATION

Factor IX Zutphen is a variant IX molecule isolated from the blood of a patient with severe haemophilia B. The molecular defect in factor IX Zutphen is a Cys(18) --> Arg mutation as a result of a T --> C transi tion at residue 6427 of the factor IX gene of the patient. The mutatio n disrupts the disulphide bond in the Gla-domain between Cys(18) and C ys(23). The remaining free cysteine residue results in the formation o f a 95 kDa complex alpha(1)-microglobulin through an intermolecular di sulphide bond. The same complex circulates at high levels in plasma of carriers of the mutation. The variant molecule has a calcium-binding defect, which is shown not to be caused by incomplete gamma-binding de fect, which is shown not to be caused by incomplete gamma-carboxylatio n. Factor IX Zutphen can not blind to phospholipids and can not be act ivated by factor XIa or by factor VIIa-tissue factor complex. Two sequ ential metal ion-dependent conformational transitions (factor IX --> f actor IX' --> factor IX) have been proposed for human factor IX [Lieb man (1987) J. Biol. Chem. 262, 7605-7612], based upon the metal ion re quirements for binding to anti-factor IX:Mg(II) antibodies, which are specific for the factor IX' conformation. We used these conformation-s pecific antibodies, and antibodies raised against a synthetic peptide corresponding to residues 35-50 of human factor IX [anti-factor IX(35- 50)] to study the metal ion-induced conformation of factor IX Zutphen. The disruption of the disulphide bond in the Gla-domain, maybe in com bination with the complex with alpha(1)-microglobulin, destabilized th e factor IX' conformation. The formation of the factor IX conformatio n was prevented independent of the prescence of alpha(1)-microglobulin . The disulphide bond in the Gla-domain is therefore essential for the calcium-dependent confirmation and function of factor IX.