Integrative phosphoproteomic analyses reveal hemostatic-endothelial signaling interplay.

IF 5.5 2区 医学 Q1 HEMATOLOGY Journal of Thrombosis and Haemostasis Pub Date : 2024-10-21 DOI:10.1016/j.jtha.2024.10.011
Stijn A Groten, Bart L van den Eshof, Floris P J van Alphen, Alexander B Meijer, Maartje van den Biggelaar, Arie J Hoogendijk
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Abstract

Background: The vascular endothelial cell (EC) monolayer plays a crucial part in maintaining hemostasis. An extensive array of G protein-coupled receptors 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 G protein-coupled receptors remains limited.

Objectives: To elucidate histamine and protease-activated receptor (PAR1-4) signaling cascades in ECs, discern overlapping and diverging regulation between these stimuli and their effect on the EC monolayer.

Methods: We employed stable isotope labeling by amino acids in cell culture mass spectrometry-based phosphoproteomics on in vitro cultured blood outgrowth ECs stimulated with histamine and different PAR1 to 4 peptides. We investigated key phosphosites through immuno(fluorescence) staining and determined effects on barrier function through transendothelial resistance assays.

Results: EC histamine activation initiated an extensive (kinase) signaling network (including MAPK3, STAT3, and CTNND1). PAR1 and PAR2 receptors induced highly similar signaling cascades, whereas PAR3 and PAR4 induced minimal phospho-regulation. Integration of all applied stimuli indicated uniquely activated proteins between both stimuli, as well as a general overlapping activation of cell junction and actin cytoskeletal proteins.

Conclusion: We provide an integrative phosphoproteomic analysis of histamine and PAR agonists in the endothelium that highlights the endothelial response programs that are at the basis of regulating hemostasis.

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综合磷酸蛋白组分析揭示了止血-内皮信号的相互作用。
背景:血管内皮细胞(EC)单层在维持止血方面发挥着至关重要的作用。一系列广泛的 G 蛋白偶联受体(GPCR)使血管内皮细胞能够动态地作用于凝血酶和组胺等关键止血刺激。这些刺激对心血管细胞信号传导的影响一直是各种研究的主题,但对不同 GPCR 之间不和谐和和谐的心血管细胞信号传导的了解仍然有限:目的:阐明组胺和蛋白酶激活受体(PAR1-4)在内皮细胞中的信号级联,辨别这些刺激之间的重叠和分歧调控及其对内皮细胞单层的影响:方法:我们采用基于氨基酸稳定同位素标记的细胞培养(SILAC)质谱技术,对组胺和不同蛋白酶激活受体肽(PAR1-4)刺激下体外培养的 BOECs 进行磷酸化蛋白质组学研究。我们通过免疫(荧光)染色研究了关键磷酸位点,并通过跨内皮阻力测定确定了对屏障功能的影响:结果:EC组胺激活启动了一个广泛的(激酶)信号网络(其中包括MAPK3、STAT3和CTNND1)。PAR1 和 PAR2 受体诱导了高度相似的信号级联,而 PAR3 和 PAR4 则诱导了最小的磷酸化调节。对所有应用刺激的整合表明,两种刺激都有独特的激活蛋白,细胞连接蛋白和肌动蛋白的激活也普遍重叠:我们对组胺和 PAR 激动剂在内皮中的作用进行了综合磷酸蛋白组学分析,突出了内皮反应程序是调节止血的基础。
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来源期刊
Journal of Thrombosis and Haemostasis
Journal of Thrombosis and Haemostasis 医学-外周血管病
CiteScore
24.30
自引率
3.80%
发文量
321
审稿时长
1 months
期刊介绍: The Journal of Thrombosis and Haemostasis (JTH) serves as the official journal of the International Society on Thrombosis and Haemostasis. It is dedicated to advancing science related to thrombosis, bleeding disorders, and vascular biology through the dissemination and exchange of information and ideas within the global research community. Types of Publications: The journal publishes a variety of content, including: Original research reports State-of-the-art reviews Brief reports Case reports Invited commentaries on publications in the Journal Forum articles Correspondence Announcements Scope of Contributions: Editors invite contributions from both fundamental and clinical domains. These include: Basic manuscripts on blood coagulation and fibrinolysis Studies on proteins and reactions related to thrombosis and haemostasis Research on blood platelets and their interactions with other biological systems, such as the vessel wall, blood cells, and invading organisms Clinical manuscripts covering various topics including venous thrombosis, arterial disease, hemophilia, bleeding disorders, and platelet diseases Clinical manuscripts may encompass etiology, diagnostics, prognosis, prevention, and treatment strategies.
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