Factor IX Zutphen is a variant
factor IX molecule isolated from the blood of a patient with severe
haemophilia B. The molecular defect in
factor IX Zutphen is a Cys18-->Arg mutation as a result of a T-->C transition at residue 6427 of the
factor IX gene of the patient. The mutation disrupts the disulphide bond in the Gla-domain between Cys18 and Cys23. The remaining free
cysteine residue results in the formation of a 95 kDa complex with alpha 1-microglobulin through an intermolecular disulphide 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-carboxylation.
Factor IX Zutphen can not bind to
phospholipids and can not be activated by
factor XIa or by
factor VIIa-
tissue factor complex. Two sequential
metal ion-dependent conformational transitions (
factor IX-->
factor IX'-->
factor IX*) have been proposed for human
factor IX [Liebman (1987) J. Biol. Chem. 262, 7605-7612], based upon the
metal ion requirements for binding to anti-
factor IX:Mg(II)
antibodies, which are specific for the
factor IX' conformation, and anti-
factor IX:Ca(II)
antibodies, which are specific for the
factor IX* conformation. We used these conformation-specific
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 combination with the complex with alpha 1-microglobulin, destabilized the
factor IX' conformation. The formation of the
factor IX* conformation was prevented independent of the presence of alpha 1-microglobulin. The disulphide bond in the Gla-domain is therefore essential for the
calcium-dependent conformation and function of
factor IX.