Nakayama Moeka, Miyagawa Hitomi, Kuranami Yumiko, Tsunooka-Ota Miyuki, Yamaguchi Yoshiki, Kojima-Aikawa Kyoko
Division of Advanced Sciences, Graduate School of Humanities and Sciences, Ochanomizu University, Tokyo, Japan.
Program for Leading Graduate Schools, Ochanomizu University, Tokyo, Japan.
J Thromb Haemost. 2020 Jun;18(6):1357-1369. doi: 10.1111/jth.14789. Epub 2020 May 6.
Factor XII (FXII) is a plasma serine protease that initiates the intrinsic pathway of blood coagulation upon contact with anionic substances, such as the sulfated glycolipid sulfatide. Annexins (ANXs) have been implicated in the regulation of the blood coagulation reaction by binding to anionic surfaces composed of phospholipids and sulfated glycoconjugates, but their physiological importance is only partially understood.
To test the hypothesis that ANXs are involved in suppressing the intrinsic pathway initiated by sulfatide, we examined the effect of eight recombinant ANX proteins on the intrinsic coagulation reaction and their sulfatide binding activities.
Recombinant ANXs were prepared in Escherichia coli expression systems and their anticoagulant effects on the intrinsic pathway initiated by sulfatide were examined using plasma clotting assay and chromogenic assay. ANXA4 active sites were identified by alanine scanning and fold deletion in the core domain.
We found that ANXA3, ANXA4, and ANXA5 strongly inhibited sulfatide-induced plasma coagulation. Wild-type and mutated ANXA4 were used to clarify the molecular mechanism involved in inhibition. ANXA4 inhibited sulfatide-induced auto-activation of FXII to FXIIa and the conversion of its natural substrate FXI to FXIa but showed no effect on the protease activity of FXIIa or FXIa. Alanine scanning showed that substitution of the Ca -binding amino acid residue in the fourth fold of the core domain of ANXA4 reduced anticoagulant activity, and deletion of the entire fourth fold of the core domain resulted in complete loss of anticoagulant activity.
凝血因子 XII（FXII）是一种血浆丝氨酸蛋白酶，在与阴离子物质（如硫酸化糖脂硫苷脂）接触时启动内源性凝血途径。膜联蛋白（ANXs）通过与由磷脂和硫酸化糖缀合物组成的阴离子表面结合，参与凝血反应的调节，但其生理重要性仅得到部分理解。
为了验证膜联蛋白参与抑制由硫苷脂引发的内源性途径这一假说，我们研究了 8 种重组膜联蛋白对内源性凝血反应的影响及其硫苷脂结合活性。
在大肠杆菌表达系统中制备重组膜联蛋白，并使用血浆凝固试验和显色试验检测它们对由硫苷脂引发的内源性途径的抗凝作用。通过丙氨酸扫描和核心结构域的折叠缺失鉴定膜联蛋白 A4（ANXA4）的活性位点。
我们发现膜联蛋白 A3（ANXA3）、膜联蛋白 A4 和膜联蛋白 A5 强烈抑制硫苷脂诱导的血浆凝固。使用野生型和突变型膜联蛋白 A4 阐明抑制作用的分子机制。膜联蛋白 A4 抑制硫苷脂诱导的 FXII 自激活为 FXIIa 及其天然底物 FXI 转化为 FXIa，但对 FXIIa 或 FXIa 的蛋白酶活性无影响。丙氨酸扫描显示，膜联蛋白 A4 核心结构域第四折叠中钙结合氨基酸残基的取代降低了抗凝活性，核心结构域整个第四折叠的缺失导致抗凝活性完全丧失。
