Francischetti Ivo M B, Valenzuela Jesus G, Andersen John F, Mather Thomas N, Ribeiro José M C
Section of Medical Entomology, Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892-0425, USA.
Blood. 2002 May 15;99(10):3602-12. doi: 10.1182/blood-2001-12-0237.
Saliva of the hard tick and Lyme disease vector, Ixodes scapularis, has a repertoire of compounds that counteract host defenses. Following sequencing of an I scapularis salivary gland complementary DNA (cDNA) library, a clone with sequence homology to tissue factor pathway inhibitor (TFPI) was identified. This cDNA codes for a mature protein, herein called Ixolaris, with 140 amino acids containing 10 cysteines and 2 Kunitz-like domains. Recombinant Ixolaris was expressed in insect cells and shown to inhibit factor VIIa (FVIIa)/tissue factor (TF)-induced factor X (FX) activation with an inhibitory concentration of 50% (IC(50)) in the picomolar range. In nondenaturing gel, Ixolaris interacted stoichiometrically with FX and FXa but not FVIIa. Ixolaris behaves as a fast-and-tight ligand of the exosites of FXa and gamma-carboxyglutamic acid domainless FXa (des-Gla-FXa), increasing its amidolytic activity. At high concentration, Ixolaris attenuates the amidolytic activity of FVIIa/TF; however, in the presence of DEGR-FX or DEGR-FXa (but not des-Gla-DEGR-FXa), Ixolaris becomes a tight inhibitor of FVIIa/TF as assessed by recombinant factor IX (BeneFIX) activation assays. This indicates that FX and FXa are scaffolds for Ixolaris in the inhibition of FVIIa/TF and implies that the Gla domain is necessary for FVIIa/TF/Ixolaris/FX(a) complex formation. Additionally, we show that Ixolaris blocks FXa generation by endothelial cells expressing TF. Ixolaris may be a useful tool to study the structural features of FVIIa, FX, and FXa, and an alternative anticoagulant in cardiovascular diseases.
硬蜱及莱姆病传播媒介肩突硬蜱的唾液含有一系列可对抗宿主防御的化合物。在对肩突硬蜱唾液腺互补DNA（cDNA）文库进行测序后，鉴定出一个与组织因子途径抑制剂（TFPI）具有序列同源性的克隆。该cDNA编码一种成熟蛋白，在此称为Ixolaris，含140个氨基酸，有10个半胱氨酸和2个Kunitz样结构域。重组Ixolaris在昆虫细胞中表达，并显示在皮摩尔范围内抑制因子VIIa（FVIIa）/组织因子（TF）诱导的因子X（FX）激活，其50%抑制浓度（IC50）在此范围。在非变性凝胶中，Ixolaris与FX和FXa化学计量地相互作用，但不与FVIIa相互作用。Ixolaris表现为FXa和无γ-羧基谷氨酸结构域的FXa（des-Gla-FXa）外位点的快速紧密配体，增加其酰胺水解活性。在高浓度时，Ixolaris减弱FVIIa/TF的酰胺水解活性；然而，通过重组因子IX（BeneFIX）激活试验评估，在存在DEGR-FX或DEGR-FXa（但不是des-Gla-DEGR-FXa）时，Ixolaris成为FVIIa/TF的紧密抑制剂。这表明FX和FXa是Ixolaris抑制FVIIa/TF的支架，意味着Gla结构域对于FVIIa/TF/Ixolaris/FX（a）复合物形成是必需的。此外，我们表明Ixolaris可阻断表达TF的内皮细胞生成FXa。Ixolaris可能是研究FVIIa、FX和FXa结构特征的有用工具，也是心血管疾病中的一种替代抗凝剂。
