Integrative phosphoproteomic analysis reveal hemostatic-endothelial signaling interplay

The vascular endothelial cell (EC) monolayer plays a crucial part in maintaining hemostasis. An extensive array of G protein-coupled receptors (GPCRs) allows ECs to dynamically act on key hemostatic stimuli such as thrombin and histamine. The impact of these individual stimuli on EC signal transduction has been the subject of various studies, but insight into discordant and concordant EC signaling between different GPCRs remain limited. Using mass spectrometry- based phosphoproteomics, we delineated the unbiased signaling response for histamine stimulation in the endothelium and observed highly similar responses between different protease activated receptors (PAR1-4). Finally, we compare both histamine and PAR agonists, indicating uniquely activated proteins between both stimuli, but a general overlapping activation of cell-junction and actin cytoskeletal proteins. Thus, we provide an integrative phosphoproteomic analysis of histamine and PAR agonist in the endothelium that highlights the endothelial response programs that are at the bases of regulating hemostasis.

血管内皮细胞（vascular endothelial cell，EC）单层在维持机体止血功能中发挥关键作用。大量G蛋白偶联受体（G protein-coupled receptors，GPCRs）使得EC能够对凝血酶、组胺等关键止血刺激物产生动态应答。目前已有多项研究探讨了单一刺激物对EC信号转导的影响，但针对不同GPCR介导的EC信号通路间的协同与拮抗应答的认知仍较为有限。本研究采用基于质谱的磷酸化蛋白质组学技术，明确了内皮细胞受组胺刺激后的无偏倚信号应答全貌，并发现不同蛋白酶激活受体（protease activated receptors，PAR1-4）介导的应答高度相似。最后，本研究对组胺与PAR激动剂进行了对比分析，结果显示两种刺激物可分别激活特异性蛋白，但二者在细胞连接蛋白与肌动蛋白细胞骨架蛋白的激活上普遍存在重叠。综上，本研究针对内皮细胞中的组胺与PAR激动剂开展了整合式磷酸化蛋白质组学分析，揭示了作为调控机体止血功能基础的内皮细胞应答程序。