Department of Clinical Medicine, Trinity College Dublin, Dublin, Ireland.
National Children's Research Centre, Our Lady's Children's Hospital Crumlin, Dublin, Ireland.
J Thromb Haemost. 2017 Nov;15(11):2198-2207. doi: 10.1111/jth.13807. Epub 2017 Sep 21.
Essentials The basis of cytoprotective protease-activated receptor 1 (PAR1) signaling is not fully understood. Activated protein C chimera (APC ) was used to identify requirements for PAR1 signaling. APC did not initiate PAR1 signaling, but conferred monocyte anti-inflammatory activity. APC-specific light chain residues are required for cytoprotective PAR1 signaling.
Background Activated protein C (APC) cell signaling is largely reliant upon its ability to mediate protease-activated receptor (PAR) 1 proteolysis when bound to the endothelial cell (EC) protein C (PC) receptor (EPCR). Furthermore, EPCR-bound PC modulates PAR1 signaling by thrombin to induce APC-like EC cytoprotection. Objective The molecular determinants of EPCR-dependent cytoprotective PAR1 signaling remain poorly defined. To address this, a PC-factor VII chimera (PC ) possessing FVII N-terminal domains and conserved EPCR binding was characterized. Methods Activated PC-FVII chimera (APC ) anticoagulant activity was measured with calibrated automated thrombography and activated FV degradation assays. APC signaling activity was characterized by the use of reporter assays of PAR1 proteolysis and EC barrier integrity. APC anti-inflammatory activity was assessed according to its inhibition of nuclear factor-κB (NF-κB) activation and cytokine secretion from monocytes. Results PC was activated normally by thrombin on ECs, but was unable to inhibit plasma thrombin generation. Surprisingly, APC did not mediate EPCR-dependent PAR1 proteolysis, confer PAR1-dependent protection of thrombin-induced EC barrier disruption, or limit PAR1-dependent attenuation of interleukin-6 release from lipopolysaccharide (LPS)-stimulated macrophages. Interestingly, EPCR occupation by active site-blocked APC was, like FVII, unable to mimic EC barrier stabilization induced by PC upon PAR1 proteolysis by thrombin. APC did, however, diminish LPS-induced NF-κB activation and tumor necrosis factor-α release from monocytes in an apolipoprotein E receptor 2-dependent manner, with similar efficacy as wild-type APC. Conclusions These findings identify a novel role for APC light chain amino acid residues outside the EPCR-binding site in enabling cytoprotective PAR1 signaling.
要点 细胞保护蛋白酶激活受体 1 (PAR1) 信号的基础尚未完全理解。使用激活蛋白 C 嵌合体 (APC) 来确定 PAR1 信号的要求。APC 本身不会启动 PAR1 信号，但赋予单核细胞抗炎活性。APC 特异性轻链残基是细胞保护 PAR1 信号所必需的。
背景 激活蛋白 C (APC) 细胞信号主要依赖于其与内皮细胞 (EC) 蛋白 C (PC) 受体 (EPCR) 结合时介导蛋白酶激活受体 (PAR) 1 蛋白水解的能力。此外，EPCR 结合的 PC 通过凝血酶诱导 APC 样 EC 细胞保护来调节 PAR1 信号。
目的 EPCR 依赖性细胞保护 PAR1 信号的分子决定因素仍未得到很好的定义。为了解决这个问题，我们对具有 FVII N 端结构域和保守 EPCR 结合的 PC-因子 VII 嵌合体 (PC ) 进行了表征。
方法 使用校准的自动血栓形成和激活 FV 降解测定法测量激活 PC-FVII 嵌合体 (APC ) 的抗凝活性。通过 PAR1 蛋白水解和 EC 屏障完整性的报告基因测定来描述 APC 信号活性。根据其对核因子-κB (NF-κB) 激活和单核细胞细胞因子分泌的抑制作用来评估 APC 的抗炎活性。
结果 PC 在 EC 上被凝血酶正常激活，但不能抑制血浆凝血酶生成。令人惊讶的是，APC 本身不能介导 EPCR 依赖性 PAR1 蛋白水解，赋予 PAR1 依赖性保护凝血酶诱导的 EC 屏障破坏，或限制 PAR1 依赖性减弱脂多糖 (LPS) 刺激的巨噬细胞释放白细胞介素-6。有趣的是，像 FVII 一样，活性位点阻断的 APC 占据 EPCR 不能模拟由凝血酶 PAR1 蛋白水解诱导的 PC 对 EC 屏障稳定性的稳定。然而，APC 确实以载脂蛋白 E 受体 2 依赖性方式减少了 LPS 诱导的单核细胞 NF-κB 激活和肿瘤坏死因子-α释放，其功效与野生型 APC 相似。
结论 这些发现确定了 APC 轻链氨基酸残基在 EPCR 结合位点之外在使细胞保护 PAR1 信号成为可能方面的新作用。
