Wojcik E G, van den Berg M, van der Linden I K, Poort S R, Cupers R, Bertina R M
Haemostasis and Thrombosis Research Centre, University Hospital, Leiden, The Netherlands.
Biochem J. 1995 Nov 1;311 ( Pt 3)(Pt 3):753-9. doi: 10.1042/bj3110753.
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.
凝血因子IX祖芬是从一名严重B型血友病患者血液中分离出的一种变异凝血因子IX分子。凝血因子IX祖芬的分子缺陷是由于患者凝血因子IX基因第6427位残基处发生T→C转换，导致Cys18突变为Arg。该突变破坏了Gla结构域中Cys18和Cys23之间的二硫键。剩余的游离半胱氨酸残基通过分子间二硫键与α1-微球蛋白形成95 kDa的复合物。在该突变携带者的血浆中，相同的复合物以高水平循环。该变异分子存在钙结合缺陷，且并非由不完全的γ-羧化作用引起。凝血因子IX祖芬不能与磷脂结合，也不能被因子XIa或因子VIIa-组织因子复合物激活。基于与抗凝血因子IX:Mg(II)抗体（对凝血因子IX'构象具有特异性）和抗凝血因子IX:Ca(II)抗体（对凝血因子IX构象具有特异性）结合所需的金属离子，有人提出人凝血因子IX存在两个连续的金属离子依赖性构象转变（凝血因子IX→凝血因子IX'→凝血因子IX）[利布曼（1987年）《生物化学杂志》262卷，7605 - 7612页]。我们使用这些构象特异性抗体以及针对与人凝血因子IX第35 - 50位残基相对应的合成肽产生的抗体[抗凝血因子IX(35 - 50)]来研究金属离子诱导的凝血因子IX祖芬的构象。Gla结构域中二硫键的破坏，可能与α1-微球蛋白复合物共同作用，使凝血因子IX'构象不稳定。无论α1-微球蛋白是否存在，凝血因子IX*构象的形成均受到阻碍。因此，Gla结构域中的二硫键对于凝血因子IX的钙依赖性构象和功能至关重要。
