Astermark J, Sottile J, Mosher D F, Stenflo J
Department of Clinical Chemistry, University of Lund, Malmö General Hospital, Sweden.
J Biol Chem. 1994 Feb 4;269(5):3690-7.
Factor IX is a vitamin K-dependent procoagulant zymogen of a serine protease. In the presence of Ca2+ the active form of factor IX (factor IXa beta) forms a complex with factor VIIIa on suitable phospholipid surfaces such as aggregated platelets. This macromolecular complex rapidly activates factor X. We have previously provided data that suggest an interaction between the NH2-terminal epidermal growth factor (EGF)-like module of factor IXa beta and the substrate factor X. In an alternative approach to study this protein-protein interaction, we have expressed three recombinant baculovirus constructs encoding the EGF-like modules of human factor IX and a truncated form of fibronectin in a system based on the infection of insect cells (Spodoptera frugiperda 21). This strategy allows a simple one-step purification of the recombinant proteins on a gelatin-Sepharose column, followed by removal of the gelatin-binding part derived from fibronectin by proteolytic cleavage. The fusion proteins were isolated at yields of 20-50 micrograms/ml culture medium. The recombinant EGF-like modules contained 0.2-0.4 mol of erythro-beta-hydroxyaspartic acid/mol of protein, i.e. similar to the amount found in factor IX from human plasma, and appeared to be glycosylated at Ser-53. The NH2-terminal EGF-like module, which contained a transamidation acceptor site derived from fibronectin, was cross-linked by factor XIIIa in solution to intact and Gla-domainless factor X. There was no evidence of cross-linking to activated factor X or to factor X fragments containing only the gamma-carboxyglutamic acid module and the two EGF-like modules. The cross-linking results suggest a specific interaction between the NH2-terminal EGF-like module of factor IXa beta and the heavy chain of unactivated factor X. This interaction, albeit weak as judged by competition experiments, may be important for the targeting of factor X to the factor IXa beta-factor VIIIa complex on biological membranes and for the subsequent dissociation of factor Xa from the complex after activation.
凝血因子IX是一种维生素K依赖的丝氨酸蛋白酶前凝血酶原。在Ca2+存在的情况下，凝血因子IX的活性形式（凝血因子IXaβ）在合适的磷脂表面（如聚集的血小板）上与凝血因子VIIIa形成复合物。这种大分子复合物迅速激活凝血因子X。我们之前提供的数据表明，凝血因子IXaβ的NH2末端表皮生长因子（EGF）样模块与底物凝血因子X之间存在相互作用。为了研究这种蛋白质-蛋白质相互作用，我们采用了另一种方法，在基于昆虫细胞（草地贪夜蛾21）感染的系统中表达了三种重组杆状病毒构建体，它们分别编码人凝血因子IX的EGF样模块和截短形式的纤连蛋白。这种策略允许在明胶-琼脂糖柱上对重组蛋白进行简单的一步纯化，然后通过蛋白水解切割去除源自纤连蛋白的明胶结合部分。融合蛋白的分离产量为20-50微克/毫升培养基。重组EGF样模块每摩尔蛋白质含有0.2-0.4摩尔的赤藓糖-β-羟基天冬氨酸，即与从人血浆中分离出的凝血因子IX中发现的量相似，并且似乎在Ser-53处进行了糖基化。含有源自纤连蛋白的转酰胺基受体位点的NH2末端EGF样模块，在溶液中被凝血因子XIIIa交联到完整的和无Gla结构域的凝血因子X上。没有证据表明它与活化的凝血因子X或仅含有γ-羧基谷氨酸模块和两个EGF样模块的凝血因子X片段发生交联。交联结果表明，凝血因子IXaβ的NH2末端EGF样模块与未活化的凝血因子X的重链之间存在特异性相互作用。尽管从竞争实验判断这种相互作用较弱，但它可能对于凝血因子X靶向生物膜上的凝血因子IXaβ-凝血因子VIIIa复合物以及激活后凝血因子Xa从复合物中随后解离很重要。
