Department of Biochemistry, Cardiovascular Research Institute Maastricht, University Maastricht, Maastricht, Netherlands.
J Thromb Haemost. 2013 Apr;11(4):704-14. doi: 10.1111/jth.12152.
Tissue factor pathway inhibitor (TFPI) is a multi-Kunitz domain protease inhibitor that down-regulates the extrinsic coagulation pathway by inhibiting FXa and FVIIa.
To investigate the role of the three Kunitz domains (KDs) of TFPI in FVIIa inhibition using full-length TFPI (TFPIfl ) and truncated TFPI constructs.
Inhibition of FVIIa with/without relipidated tissue factor (TF) or soluble TF (sTF) by TFPIfl /TFPI constructs was quantified with a FVIIa-specific chromogenic substrate.
TFPIfl inhibited TF-FVIIa via a monophasic reaction, which is rather slow at low TFPIfl concentrations (t½ ≈ 5 min at 2 nm TFPI) and has a Ki of 4.6 nm. In the presence of sTF and without TF, TFPIfl was a poor FVIIa inhibitor, with Ki values of 122 nm and 1118 nm, respectively. This indicates that phospholipids and TF significantly contribute to FVIIa inhibition by TFPIfl . TFPI constructs without the KD3-c-terminus (TFPI1-150 and KD1-KD2) were 7-10-fold less effective than TFPIfl in inhibiting TF-FVIIa and sTF-FVIIa, indicating that the KD3-C-terminus significantly contributes to direct inhibition of FVIIa by TFPI. Compared with KD1-KD2, KD1 was a poor TF-FVIIa inhibitor (Ki =434 nm), which shows that the KD2 domain of TFPI also contributes to FVIIa inhibition. Protein S stimulated TF-FVIIa inhibition by TFPIfl (Ki =0.7 nm). In the presence of FXa, a tight quaternary TF-FVIIa-TFPI-FXa complex is formed with TFPIfl , TFPI1-150 and KD1-KD2, with Ki values of < 0.15 nm, 0.5 nm and 0.8 nm, respectively, indicating the KD3-C-terminus is not a prerequisite for quaternary complex formation. Phospholipids and the Gla-domain of FXa are required for quaternary complex formation.
组织因子途径抑制剂（TFPI）是一种多 Kunitz 结构域蛋白酶抑制剂，通过抑制 FXa 和 FVIIa 来下调外源性凝血途径。
使用全长 TFPI（TFPIfl）和截断 TFPI 构建体研究 TFPI 的三个 Kunitz 结构域（KDs）在 FVIIa 抑制中的作用。
用 FVIIa 特异性显色底物定量测定 TFPIfl/TFPI 构建体对有/无再脂化组织因子（TF）或可溶性 TF（sTF）的 FVIIa 抑制作用。
TFPIfl 通过单相反应抑制 TF-FVIIa，在低 TFPIfl 浓度下反应相当缓慢（在 2nm TFPI 时 t½≈5min），Ki 为 4.6nm。在 sTF 存在且无 TF 时，TFPIfl 对 FVIIa 的抑制作用较差，Ki 值分别为 122nm 和 1118nm，这表明磷脂和 TF 显著促进 TFPIfl 对 FVIIa 的抑制作用。无 KD3-C 末端的 TFPI 构建体（TFPI1-150 和 KD1-KD2）在抑制 TF-FVIIa 和 sTF-FVIIa 方面比 TFPIfl 有效 7-10 倍，表明 KD3-C 末端显著促进 TFPI 对 FVIIa 的直接抑制。与 KD1-KD2 相比，KD1 是一种较差的 TF-FVIIa 抑制剂（Ki=434nm），这表明 TFPI 的 KD2 结构域也有助于 FVIIa 的抑制。蛋白 S 刺激 TFPIfl 对 TF-FVIIa 的抑制作用（Ki=0.7nm）。在 FXa 存在下，TFPIfl、TFPI1-150 和 KD1-KD2 形成紧密的四元 TF-FVIIa-TFPI-FXa 复合物，Ki 值分别<0.15nm、0.5nm 和 0.8nm，表明 KD3-C 末端不是形成四元复合物的必要条件。磷脂和 FXa 的 Gla 结构域是形成四元复合物所必需的。
