Department of Biochemistry, Cardiovascular Research Institute Maastricht, Maastricht University, the Netherlands.
Department of Human Biology, NUTRIM School of Nutrition and Translational Research in Metabolism, Maastricht University Medical Center, Maastricht, the Netherlands.
J Thromb Haemost. 2023 May;21(5):1214-1226. doi: 10.1016/j.jtha.2023.01.017. Epub 2023 Jan 28.
Tissue factor pathway inhibitor (TFPI) is an important regulator of coagulation and a link between inflammation and thrombosis. During thrombotic events, TFPI is proteolytically inactivated by neutrophil elastase while bound to neutrophil extracellular traps (NETs). Protein arginine deiminase 4 (PAD4) catalyzes the conversion of arginine to citrulline and is crucial for NET formation.
Here, we show that PAD4 inactivates full-length TFPIα by citrullination of its functional arginines.
Citrullination of TFPIα and of TFPI-constructs by PAD4 was studied using western blotting and mass spectrometry. Binding of TFPIα to PAD4 was investigated using a solid-phase assay. Functional consequences were investigated by factor Xa inhibition and thrombin generation assays.
Nanomolar PAD4 amounts eliminated factor Xa inhibition by TFPIα. A citrullinated mutant Kunitz 2 domain did not inhibit factor Xa. Citrullination of TFPIα was found to be time- and concentration-dependent. Immunoprecipitation of citrullinated proteins from whole blood after neutrophil activation suggested the presence of TFPIα. Negatively charged phospholipids inhibited citrullination and truncated variants K1K2 and TFPI 1-161, and the isolated K2 domain were less efficiently citrullinated by PAD4. TFPIα bound to PAD4 with nanomolar affinity and involved the basic C-terminus. Thrombin generation in TFPI-deficient plasma demonstrated reduced anticoagulant activity of citrullinated TFPI. Mass spectrometry demonstrated citrullination of surface-exposed arginine residues in TFPIα after incubation with PAD4.
Full-length TFPIα is sensitive to citrullination by PAD4, which causes loss of factor Xa inhibition. This process may play a role in the increased thrombosis risk associated with inflammation.
组织因子途径抑制剂（TFPI）是凝血的重要调节剂，也是炎症与血栓形成之间的联系。在血栓形成事件中，TFPI 与中性粒细胞胞外诱捕网（NETs）结合时会被中性粒细胞弹性蛋白酶蛋白水解失活。蛋白精氨酸脱亚氨酶 4（PAD4）催化精氨酸向瓜氨酸的转化，对 NET 的形成至关重要。
本研究旨在表明 PAD4 通过精氨酸的瓜氨酸化使全长 TFPIα失活。
使用 Western blot 和质谱法研究了 PAD4 对 TFPIα 和 TFPI 构建体的瓜氨酸化作用。使用固相测定法研究了 TFPIα 与 PAD4 的结合。通过 Xa 因子抑制和凝血酶生成测定研究了功能后果。
纳米摩尔 PAD4 量可消除 TFPIα 对 Xa 因子的抑制作用。不抑制 Xa 因子的瓜氨酸化突变 Kunitz 2 结构域。发现 TFPIα 的瓜氨酸化作用具有时间和浓度依赖性。中性粒细胞激活后从全血中免疫沉淀的瓜氨酸化蛋白表明存在 TFPIα。带负电荷的磷脂抑制瓜氨酸化，截短变体 K1K2 和 TFPI 1-161，以及分离的 K2 结构域更不易被 PAD4 瓜氨酸化。TFPIα 与 PAD4 以纳摩尔亲和力结合，并涉及基本的 C 末端。TFPI 缺陷型血浆中的凝血酶生成表明瓜氨酸化 TFPI 的抗凝活性降低。质谱法表明，TFPIα 在与 PAD4 孵育后，表面暴露的精氨酸残基发生瓜氨酸化。
全长 TFPIα 易受 PAD4 的瓜氨酸化作用影响，从而导致 Xa 因子抑制作用丧失。该过程可能在与炎症相关的血栓形成风险增加中起作用。
