Factor IXHollywood: substitution of Pro55 by Ala in the first epidermal growth factor-like domain.

Factor IX is a multidomain protein essential for hemostasis. We describe a mutation in a patient affecting the first epidermal growth factor (EGF)-like domain of the protein. All exons and the promoter region of the gene were amplified by the polymerase chain reaction method, and sequenced. Only a single mutation (C----G), that predicts the substitution of Pro55 by Ala in the first EGF domain was found in the patient's gene. This mutation leads to new restriction sites for four enzymes. One new site (Nsi) was tested in the amplified exon IV fragment and was shown to provide a rapid and reliable marker for carrier detection and prenatal diagnosis in the affected family. The factor IX protein, termed factor IXHollywood (IXHW), was isolated to homogeneity from the patient's plasma. As compared with normal factor IX (IXN), IXHW contained the same amount of gamma-carboxy glutamic acid but twice the amount of beta-OH aspartic acid. Both IXHW and IXN contained no detectable free -SH groups. Further, IXHW could be readily cleaved to yield a factor IXa-like molecule by factor Xla/Ca2+. However, IXaHW (compared with IXaN) activated factor X approximately twofold slower in the presence of Ca2+ and phospholipid (PL), and 8- to 12-fold slower in the presence of Ca2+, PL, and factor VIIIa. Additionally, IXaHW had only approximately 10% of the activity of IXaN in an aPTT assay. In agreement with the nuclear magnetic resonance-derived structure of EGF, the Chou-Fasman algorithm strongly predicted a beta turn involving residues Asn-Pro55-Cys-Leu in IXN. Replacement of Pro55 by Ala gave a fourfold decrease in the beta turn probability for this peptide, suggesting a change(s) in the secondary structure in the EGF domain of IXHW. Since this domain of IXN is thought to have one high-affinity Ca2+ binding site and may be involved in PL and/or factor VIIIa binding, the localized secondary structural changes in IXHW could lead to distortion of the binding site(s) for the cofactor(s) and, thus, a dysfunctional molecule.