Iyer Abishek, Humphries Tyrone L R, Owens Evan P, Zhao Kong-Nan, Masci Paul P, Johnson David W, Nikolic-Paterson David, Gobe Glenda C, Fairlie David P, Vesey David A
Australian Research Council Centre of Excellence in Advanced Molecular Imaging, Institute for Molecular Bioscience, The University of Queensland, Brisbane, QLD, Australia.
Centre for Inflammation and Disease Research, Institute for Molecular Bioscience, The University of Queensland, Brisbane, QLD, Australia.
Front Physiol. 2021 Mar 10;12:615428. doi: 10.3389/fphys.2021.615428. eCollection 2021.
Coagulation abnormalities and increased risk of atherothrombosis are common in patients with chronic kidney diseases (CKD). Mechanisms that alter renal hemostasis and lead to thrombotic events are not fully understood. Here we show that activation of protease activated receptor-2 (PAR2) on human kidney tubular epithelial cells (HTECs), induces tissue factor (TF) synthesis and secretion that enhances blood clotting. PAR-activating coagulation-associated protease (thrombin), as well as specific PAR2 activators (matriptase, trypsin, or synthetic agonist 2f-LIGRLO-NH (2F), induced TF synthesis and secretion that were potently inhibited by PAR2 antagonist, I-191. Thrombin-induced TF was also inhibited by a PAR1 antagonist, Vorapaxar. Peptide activators of PAR1, PAR3, and PAR4 failed to induce TF synthesis. Differential centrifugation of the 2F-conditoned medium sedimented the secreted TF, together with the exosome marker ALG-2 interacting protein X (ALIX), indicating that secreted TF was associated with extracellular vesicles. 2F-treated HTEC conditioned medium significantly enhanced blood clotting, which was prevented by pre-incubating this medium with an antibody for TF. In summary, activation of PAR2 on HTEC stimulates synthesis and secretion of TF that induces blood clotting, and this is attenuated by PAR2 antagonism. Thrombin-induced TF synthesis is at least partly mediated by PAR1 transactivation of PAR2. These findings reveal how underlying hemostatic imbalances might increase thrombosis risk and subsequent chronic fibrin deposition in the kidneys of patients with CKD and suggest PAR2 antagonism as a potential therapeutic strategy for intervening in CKD progression.
凝血异常和动脉粥样硬化血栓形成风险增加在慢性肾脏病（CKD）患者中很常见。改变肾脏止血并导致血栓形成事件的机制尚未完全明确。在此我们表明，人肾小管上皮细胞（HTECs）上蛋白酶激活受体-2（PAR2）的激活会诱导组织因子（TF）的合成和分泌，从而增强血液凝固。PAR激活的凝血相关蛋白酶（凝血酶）以及特定的PAR2激活剂（matriptase、胰蛋白酶或合成激动剂2f-LIGRLO-NH（2F））诱导的TF合成和分泌被PAR2拮抗剂I-191有效抑制。凝血酶诱导的TF也被PAR1拮抗剂沃拉帕沙抑制。PAR1、PAR3和PAR4的肽激活剂未能诱导TF合成。对2F条件培养基进行差速离心可沉淀分泌的TF，以及外泌体标志物ALG-2相互作用蛋白X（ALIX），表明分泌的TF与细胞外囊泡相关。2F处理的HTEC条件培养基显著增强血液凝固，而用TF抗体预孵育该培养基可防止这种情况。总之，HTEC上PAR2的激活刺激了诱导血液凝固的TF的合成和分泌，而PAR2拮抗作用可减弱这种作用。凝血酶诱导的TF合成至少部分由PAR2的PAR1反式激活介导。这些发现揭示了潜在的止血失衡如何可能增加CKD患者肾脏中血栓形成风险和随后的慢性纤维蛋白沉积，并表明PAR2拮抗作用作为干预CKD进展的潜在治疗策略。
