MODIFICATION OF THE N-TERMINUS OF HUMAN FACTOR-IX BY DEFECTIVE PROPEPTIDE CLEAVAGE OR ACETYLATION RESULTS IN A DESTABILIZED CALCIUM-INDUCEDCONFORMATION - EFFECTS ON PHOSPHOLIPID-BINDING AND ACTIVATION BY FACTOR XIA

The propeptide of human coagulation factor IX (FIX) directs the gamma- carboxylation of the first 12 glutamic acid residues of the mature pro tein into gamma-carboxyglutamic acid (Gla) residues. The propeptide is normally removed before secretion of FIX into the blood. However, mut ation of Arg(-4) in the propeptide abolishes propeptide cleavage and r esults in circulating prefactor IX in the blood. We studied three such genetic variants, factor IX Boxtel (Arg(-4) --> Trp), factor IX Bendo rf (Arg(-4) --> Leu) and factor IX Seattle C (Arg(-4) --> Gln). These variant prefactor IX molecules bind normally to anti-FIX:Mg(II) antibo dies, which indicates that the mutations do not seriously affect gamma -carboxylation. Metal ion titration of the binding of variant prefacto r IX to conformation-specific antibodies demonstrates that the calcium -induced conformation is destabilized in the variant molecules. Also t he binding of FIX Boxtel to phospholipids and its activation by factor XIa requires a high (>5 mM) calcium concentration. The three-dimensio nal structure of the Gla domain of FIX in the presence of calcium indi cates that the acylation of the amino-terminus, rather than the presen ce of the propeptide, was responsible for the destabilization of the c alcium-induced conformation. In order to confirm this, the alpha-amino group of Tyr(1) of FIX was acetylated. This chemically modified FIX s howed a similar destabilization of the calcium-induced conformation to variant prefactor IX. Our data imply that the amino-terminus of FIX p lays an important role in stabilizing the calcium-induced conformation of the Gla domain of FIX. This conformation is important for the bind ing to phospholipids as well as for the activation by factor XIa. Our results indicate that mutations in FIX that interfere with propeptide cleavage affect the function of the protein mainly by destabilizing th e calcium-induced conformation.