A Thrombin-PAR1/2 Feedback Loop Amplifies Thrombo-Inflammatory Endothelial Responses To The Viral RNA Analogue Poly(I:C)
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE171228 | GEO | 2021/04/01
REPOSITORIES: GEO
Similar Datasets
Project description:A Thrombin-PAR1/2 Feedback Loop Amplifies Thrombo-Inflammatory Endothelial Responses To The Viral RNA Analogue Poly(I:C)
| PRJNA718831 | ENA
Project description:Phospho-proteomics of biased PAR1 signaling induced by thrombin and APC in human cultured endothelial cells
2019-11-20 | MSV000084604 | MassIVE
Project description:Objective: Thrombin is the key serine protease of the coagulation cascade and mediates cellular responses by activation of protease-activated receptors (PARs). The predominant thrombin receptor is PAR1 and in endothelial cells (ECs) thrombin dynamically regulates a plethora of phosphorylation events. However, it has remained unclear if thrombin signaling is exclusively mediated through PAR1. Furthermore, mechanistic insight into activation and inhibition of PAR1-mediated EC signaling is lacking. In addition, signaling networks of biased PAR1 activation after differential cleavage of the PAR1 N-terminus have remained an unresolved issue.Approach and Results: Here, we used a quantitative phosphoproteomics approach to show that ‘classical’ and ‘peptide’ activation of PAR1 induce highly similar signaling, that low thrombin concentrations initiate only limited phosphoregulation, and that the PAR1 inhibitors vorapaxar and parmodulin-2 demonstrate distinct antagonistic properties. Subsequent analysis of the thrombin-regulated phosphosites in presence of PAR1 inhibitors revealed that biased activation of PAR1 is not solely linked to a specific G-protein downstream of PAR1. In addition, we showed that only the canonical thrombin PAR1 tethered ligand induces extensive early phosphoregulation in ECs.Conclusions: Our study provides detailed insight in the signaling mechanisms downstream of PAR1. Our data demonstrates that thrombin-induced EC phosphoregulation is mediated exclusively through PAR1, that thrombin and thrombin-TL peptide induce similar phosphoregulation and that only canonical PAR1 cleavage by thrombin generates a tethered ligand that potently induces early signaling. Furthermore, platelet PAR1 inhibitors directly affect EC signaling, indicating it will be a challenge to design a PAR1 antagonist that will target only those pathways responsible for tissue pathology.
2017-08-22 | PXD004264 | Pride
Project description:Thrombin is the key serine protease of the coagulation cascade and a potent trigger of protease-activated receptor (PAR)1-mediated platelet aggregation. In recent years, PAR1 has become an appealing target for anticoagulant therapies. However, the inhibitors that have been developed so far increase bleeding risk in patients, likely because they interfere with endogenous PAR1 signaling in the endothelium. Due to its complexity, thrombin-induced signaling in endothelial cells has remained incompletely understood. Here, we have combined stable isotope amino acids in cell culture, affinity-based phospho-peptide enrichment and high resolution mass spectrometry and performed a time-resolved analysis of the thrombin-induced signaling in human primary endothelial cells. We identified 2224 thrombin-regulated phosphorylation sites, the majority of which has not been previously related to thrombin. Those sites were localized on proteins which are novel to thrombin signaling, but also on well-known players such as PAR1, Rho-associated kinase 2, phospholipase C, and proteins related to actin cytoskeleton, cell-cell junctions and Weibel-Palade body release. Our study provides a unique resource of phosphoproteins and phosphorylation sites which may generate novel insights into an intimate understanding of thrombin-mediated PAR signaling and the development of improved PAR1 antagonists that affect platelet but not endothelial cell function.
2014-02-06 | PXD000597 | Pride
Project description: Not available
| S-EPMC8288670 | biostudies-literature
Project description:Transcription profiling of wild type and transgenic 35S:PAR1-GG seedlings. To characterize the transcriptional networks altered by constitutive overexpression of PAR1 in seedlings, using two-color long-oligonucleotide microarrays.
2007-11-03 | E-ATMX-29 | biostudies-arrayexpress
Project description:shRNA against PAR1 and control haripin sh004 transduced PANC-1(Pancreatic cancer) cell line
2021-05-14 | GSE155010 | GEO
Project description:Confluent human umbilical vein endothelial cells (HUVECs) were exposed to Thrombin (2 U/mL) for 2 hours. Ribosomal profiling via gradient centrifugation and fractionation was used to separate monosome, or under-translated, and polysome, or actively translated, mRNA species that were then used to probe cDNA arrays, a process known as Translation State Array Analysis (TSAA). Four samples were obtained from these experiments, Control Monosome, Control Polysome, Thrombin Monosome, and Thrombin Polysome. Using the normalized signal intensities from the GeneFilters, we calculated a translation index, or measure of movement of an mRNA molecule from the monosome to the polysome fraction upon stimulation. This calculation was made as follows: (thrombin polysome/thrombin monosome)/(control polysome/control monosome). Translational indices greater than 2.5 (upregulated) or lower than 0.4 (downregulated) were chosen for further study. TSAA data suggests that JunB is translationally regulated by thrombin stimulation. Immunocytochemistry, western blotting and RT-PCR were used to verify the results of TSAA. Keywords: Translation State Array Analysis
2006-05-31 | GSE4919 | GEO
Project description:Kallikrein-related peptidase 6 (KLK6) is a secreted serine protease hypothesized to promote inflammation via cleavage of protease-activated receptors (PAR)1 and PAR2. KLK6 levels are elevated in multiple inflammatory and autoimmune conditions, but no definitive role in pathogenesis has been established. Here, we show that skin-targeted overexpression of KLK6 causes generalized, severe psoriasiform dermatitis with spontaneous development of debilitating psoriatic arthritis-like joint disease. The psoriatic skin and joint phenotypes are reversed by normalization of skin KLK6 levels and attenuated following genetic elimination of PAR1 but not PAR2. Conservation of this regulatory pathway was confirmed in human psoriasis using vorapaxar, an FDA-approved PAR1 antagonist, on explanted lesional skin from psoriasis patients. Beyond defining a critical role for KLK6-PAR1 signaling in promoting psoriasis, our results demonstrate that KLK6-PAR1-mediated inflammation in the skin alone is sufficient to drive inflammatory joint disease. Further, we identify PAR1 as a promising cytokine-independent target in therapy of psoriasis and psoriatic arthritis.
2020-03-25 | GSE144312 | GEO
Project description:Factor XI (FXI) is a protein in the intrinsic coagulation pathway that can be activated by two enzymes. In hemostasis, FXI is activated by thrombin, while FXIIa-mediated activation of FXI is thought to be prothrombotic. The interactions of these enzymes with FXI are poorly understood due to their transient nature which is difficult to study. Here, we applied crosslinking mass spectrometry (XLMS) and molecular dynamics simulations to investigate the binding interface between thrombin and FXI as well as the dynamics underlying the activation of FXI. From the experimental data we were able to construct the FXI homodimer as well as locate the binding interface of thrombin on the light chain of FXI. As this placed thrombin distal from both the activation site as well as the apple 1 domain that thrombin is known to interact with, we applied molecular dynamics simulations to investigate the chain of events after binding. From the resulting model, we hypothesize that the activation of FXI is a multi-staged procedure where thrombin first binds to Pro520 on FXI, after which it migrates towards the activation site by first engaging the apple 1 domain and finally Arg378. We validated the results with known mutation sites on FXI and additionally found that Pro520 is conserved in PK and enables binding of thrombin to this enzyme even though it cannot be activated by it. This interaction points a way for future interventions for bleeding and thrombosis.
2024-02-05 | PXD039489 | Pride