Global Research, Novo Nordisk A/S, Måløv, Denmark.
Novo Nordisk Research Center China, Beijing, China.
J Thromb Haemost. 2018 May;16(5):893-904. doi: 10.1111/jth.14000. Epub 2018 Apr 6.
Essentials Activated FVII (FVIIa) and FX (FXa) are inhibited by tissue factor pathway inhibitor (TFPI). A monoclonal antibody, mAb2F22, was raised against the N-terminal fragment of TFPI (1-79). mAb2F22 bound exclusively to the K1 domain of TFPI (K ∼1 nm) and not to the K2 domain. mAb2F22 interfered with inhibition of both FVIIa and FXa activities and restored clot formation.
Background Initiation of coagulation is induced by binding of activated factor VII (FVIIa) to tissue factor (TF) and activation of factor X (FX) in a process regulated by tissue factor pathway inhibitor (TFPI). TFPI contains three Kunitz-type protease inhibitor domains (K1-K3), of which K1 and K2 block the active sites of FVIIa and FXa, respectively. Objective To produce a monoclonal antibody (mAb) directed towards K1, to characterize the binding epitope, and to study its effect on TFPI inhibition. Methods A monoclonal antibody, mAb2F22, was raised against the N-terminal TFPI(1-79) fragment. Binding data were obtained by surface plasmon resonance analysis. The Fab-fragment of mAb2F22, Fab2F22, was expressed and the structure of its complex with TFPI(1-79) determined by X-ray crystallography. Effects of mAb2F22 on TFPI inhibition were measured in buffer- and plasma-based systems. Results mAb2F22 bound exclusively to K1 of TFPI (K ~1 nm) and not to K2. The crystal structure of Fab2F22/TFPI (1-79) mapped an epitope on K1 including seven residues upstream of the domain. TFPI inhibition of TF/FVIIa amidolytic activity was neutralized by mAb2F22, although the binding epitope on K1 did not include the P1 residue. Binding of mAb2F22 to K1 blocked TFPI inhibition of the FXa amidolytic activity and normalized hemostasis in hemophilia human A-like plasma and whole blood. Conclusion mAb2F22 blocked TFPI inhibition of both FVIIa and FXa activities and mapped a FXa exosite for binding to K1. It reversed TFPI feedback inhibition of TF/FVIIa-induced coagulation and restored clot formation in FVIII-neutralized human plasma and blood.
凝血的启动是由激活的因子 VII（FVIIa）与组织因子（TF）结合以及因子 X（FX）在组织因子途径抑制剂（TFPI）调节下的激活引起的。TFPI 包含三个 Kunitz 型蛋白酶抑制剂结构域（K1-K3），其中 K1 和 K2 分别阻断 FVIIa 和 FXa 的活性位点。
针对 N 端 TFPI（1-79）片段产生了一种单克隆抗体（mAb），mAb2F22。通过表面等离子体共振分析获得结合数据。mAb2F22 的 Fab 片段，Fab2F22，被表达，并通过 X 射线晶体学确定其与 TFPI（1-79）复合物的结构。在缓冲液和血浆基系统中测量 mAb2F22 对 TFPI 抑制的影响。
mAb2F22 仅与 TFPI 的 K1 结合（K ~1nm），而不与 K2 结合。Fab2F22/TFPI（1-79）的晶体结构映射了 K1 上的一个表位，包括该结构域上游的七个残基。尽管 K1 上的结合表位不包括 P1 残基，但 mAb2F22 中和了 TFPI 对 TF/FVIIa 氨肽酶活性的抑制。mAb2F22 与 K1 的结合阻断了 TFPI 对 FXa 氨肽酶活性的抑制，并使血友病 A 样人血浆和全血中的止血正常化。
mAb2F22 阻断了 TFPI 对 FVIIa 和 FXa 活性的抑制，并绘制了 FXa 结合 K1 的外位。它逆转了 TFPI 对 TF/FVIIa 诱导的凝血的反馈抑制，并在 FVIII 中和的人血浆和血液中恢复了血栓形成。
