Journal: Journal of Negative Results in Biomedicine

Article Title: Interference with protease-activated receptor 1 does not reduce damage to subventricular zone cells of immature rodent brain following exposure to blood or blood plasma

doi: 10.1186/s12952-014-0022-4

Figure Lengend Snippet: Bar graph showing effects of PAR1 inhibitors on rat oligodendrocyte precursor cells (OPC) cultured in serum-free defined media. Cell death was measured by LDH release (optical density units; mean ± SEM). At high concentrations both SCH79797 and BMS20061 were toxic to the cells (*p < 0.05).

Article Snippet: Four hours later the media was changed to serum-free Sato’s medium with added 10 μM bromodeoxyuridine (BrdU Sigma B-5002) with or without added PAR1 antagonists SCH-79797 dihydrochloride (Tocris, Ellisville, MO, USA) or BMS-200261 (Sigma).

Techniques: Cell Culture

Journal: Journal of Negative Results in Biomedicine

Article Title: Interference with protease-activated receptor 1 does not reduce damage to subventricular zone cells of immature rodent brain following exposure to blood or blood plasma

doi: 10.1186/s12952-014-0022-4

Figure Lengend Snippet: Bar graph showing effects of plasma alone and in combination with PAR1 inhibitors on rat oligodendrocyte precursor cells (OPC) in culture. Cell death was measured by LDH release (optical density units; mean ± SEM). Plasma at 1/250 concentration added to the defined culture media was toxic to OPC as indicated by increased LDH release. There was no evidence for protection with SCH79797 and BMS20061 at any dose. Control values (no plasma) were significantly less than all groups (*p < 0.05) and there were no statistically significant differences between treatment groups (all comparisons p > 0.5).

Article Snippet: Four hours later the media was changed to serum-free Sato’s medium with added 10 μM bromodeoxyuridine (BrdU Sigma B-5002) with or without added PAR1 antagonists SCH-79797 dihydrochloride (Tocris, Ellisville, MO, USA) or BMS-200261 (Sigma).

Techniques: Clinical Proteomics, Concentration Assay, Control

Journal: Journal of Negative Results in Biomedicine

Article Title: Interference with protease-activated receptor 1 does not reduce damage to subventricular zone cells of immature rodent brain following exposure to blood or blood plasma

doi: 10.1186/s12952-014-0022-4

Figure Lengend Snippet: Cell proliferation in immature mouse brain following periventricular blood injection. A - Ki67 nuclear immunoreactivity (brown stained nuclei; hematoxylin counterstain) is prominent in the frontal periventricular subventricular zone (SVZ, arrow) of control (i.e. no blood injection) mice from all three genotypes (this example is wild type). B – Following blood injection, some of which extends into the frontal horn of the lateral ventricle (arrow), Ki67 immunoreactivity in the SVZ is reduced (this example is a PAR1 knockout). C – Quantitative analysis (ANOVA) shows the proportion of Ki67 immunoreactive cells in the SVZ was significantly reduced in the 24-hour heterozygous (Het; *p = 0.0050) and the 48-hour wild type (WT; *p = 0.0054) mice; the reduction also approached significance in the 48-hour knockout (KO; p = 0.0577) in comparison to controls. Bar = 100 μm.

Article Snippet: Four hours later the media was changed to serum-free Sato’s medium with added 10 μM bromodeoxyuridine (BrdU Sigma B-5002) with or without added PAR1 antagonists SCH-79797 dihydrochloride (Tocris, Ellisville, MO, USA) or BMS-200261 (Sigma).

Techniques: Injection, Staining, Control, Knock-Out, Comparison

Journal: Journal of Negative Results in Biomedicine

Article Title: Interference with protease-activated receptor 1 does not reduce damage to subventricular zone cells of immature rodent brain following exposure to blood or blood plasma

doi: 10.1186/s12952-014-0022-4

Figure Lengend Snippet: Cell death in immature mouse brain following periventricular blood injection. A - Activated caspase 3 immunoreactivity is extremely rare in the SVZ of control mice (brown stained cells; hematoxylin counterstain) (arrow; this example is wild type). B – Following blood injection, activated caspase 3 immunoreactivity is prominent in the striatum (arrow) but very rare in the SVZ (this example is a PAR1 knockout). C – Quantitative analysis shows a statistically significant increase in caspase 3 immunoreactive cells in the striatum of most groups in comparison to intact control (p < 0.007), but there were no significant differences between the blood-injected groups (all p > 0.15; Wilcoxon method). All micrographs were taken at 400× magnification. Bar = 100 μm.

Article Snippet: Four hours later the media was changed to serum-free Sato’s medium with added 10 μM bromodeoxyuridine (BrdU Sigma B-5002) with or without added PAR1 antagonists SCH-79797 dihydrochloride (Tocris, Ellisville, MO, USA) or BMS-200261 (Sigma).

Techniques: Injection, Control, Staining, Knock-Out, Comparison

Journal: PLoS Neglected Tropical Diseases

Article Title: Echis carinatus snake venom metalloprotease-induced toxicities in mice: Therapeutic intervention by a repurposed drug, Tetraethyl thiuram disulfide (Disulfiram)

doi: 10.1371/journal.pntd.0008596

Figure Lengend Snippet: Neutrophils were pre-sensitized with selective antagonists of ERK (U0126), PAR-1 (SCH79797) and PAR-2 (GB-83) for 15 min, separately. Pre-sensitized neutrophils were stimulated with 25 μg of ECV for 180 min and cells were stained with Hoechst stain. Neutrophils were photographed under a microscope (A) and, NETs were quantitated and represented as percent NETosis (B) . The data represented as mean ± SEM. *p < 0.05, when compared ECV versus ECV + antagonists. The whole cell lysates were analyzed for the phosphorylated ERK, expression of PAD4 and citH3 using Western blotting (C) . The p-ERK and PAD4 bands were quantitated using β-actin as a loading control. The citH3 bands were quantitated using H3 as a loading control. Data are representative of two independent experiments.

Article Snippet: SCH79797 (PAR-1 antagonist) and GB-83 (PAR-2 antagonist) were purchased from Cayman Chemicals (Michigan, USA).

Techniques: Staining, Microscopy, Expressing, Western Blot

Journal: PLoS Neglected Tropical Diseases

Article Title: Echis carinatus snake venom metalloprotease-induced toxicities in mice: Therapeutic intervention by a repurposed drug, Tetraethyl thiuram disulfide (Disulfiram)

doi: 10.1371/journal.pntd.0008596

Figure Lengend Snippet: Mice footpads (n = 5) were pre-treated without or with PAR-1 antagonist (SCH79797) for 15 min and followed by the injection of ECV (½LD 50 ; 1.10 mg/kg). Mice footpads were photographed from day 1 to day 8 (A) and tissue injury was measured manually on a scale of 1 to 5 (B) . Red arrow indicates edema and black arrow indicates tissue necrosis. Data are representative of two independent experiments.

Article Snippet: SCH79797 (PAR-1 antagonist) and GB-83 (PAR-2 antagonist) were purchased from Cayman Chemicals (Michigan, USA).

Techniques: Injection

Journal: Journal of Neuroinflammation

Article Title: Thrombin acts as inducer of proinflammatory macrophage migration inhibitory factor in astrocytes following rat spinal cord injury

doi: 10.1186/s12974-022-02488-w

Figure Lengend Snippet: Determination of thrombin, PARs, and MIF expression at lesion sites following rat SCI. a ELISA measurement of thrombin protein levels at lesion sites following SCI at 0, 1, 4, and 7d. b Western blot analysis of MIF following SCI at 0, 1, 4, and 7d. Quantities were normalized to endogenous β-actin. c PCR assay for determining the expression abundance of par1 , par3, and par4 in the intact spinal cord. Quantities were normalized to endogenous gapdh . d–f RT-PCR assays of par1 ( d ), par3 ( e ), and par4 ( f ) transcriptional changes following SCI at 0, 1, 4, and 7d, respectively. Quantities were normalized to endogenous gapdh . Experiments were performed at least in technical triplicates. The values shown in the figures were the average of each technical replicate. Error bars represent the SEM (* P < 0.05). g Colocalization of PAR1 with GFAP-positive astrocytes before and after SCI. The sections were prepared using a cryostat from 0.25 cm length to the lesion epicenter. Rectangle indicates region magnified. Arrowheads indicate the positive signals. Scale bars, 500 μm, and 50 μm in magnification

Article Snippet: For drug delivery, a total of 4.5 μl of 5 mM PAR1 inhibitor SCH79797 (R&D systems) was slowly injected intrathecally, prior to the incision suture.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Western Blot, Reverse Transcription Polymerase Chain Reaction

Journal: Journal of Neuroinflammation

Article Title: Thrombin acts as inducer of proinflammatory macrophage migration inhibitory factor in astrocytes following rat spinal cord injury

doi: 10.1186/s12974-022-02488-w

Figure Lengend Snippet: Functional annotation of DEGs following astrocyte stimulation with thrombin. a Primary cultured rat spinal cord astrocytes stained with GFAP and Hoechst 33,342 with purity over 95%. b PCR assay for determining the abundance of par1 , par3, and par4 at transcriptional levels in the primary astrocytes. Quantities were normalized to endogenous gapdh . c Bar graphs of DEGs following astrocyte stimulation with 100 nM thrombin for 6, 12, and 24 h, respectively. d Integration of DEGs at 6, 12, and 24 h. e GO analysis of the DEGs relating to biological processes. Scale bar, 50 μm in a

Article Snippet: For drug delivery, a total of 4.5 μl of 5 mM PAR1 inhibitor SCH79797 (R&D systems) was slowly injected intrathecally, prior to the incision suture.

Techniques: Functional Assay, Cell Culture, Staining

Journal: Journal of Neuroinflammation

Article Title: Thrombin acts as inducer of proinflammatory macrophage migration inhibitory factor in astrocytes following rat spinal cord injury

doi: 10.1186/s12974-022-02488-w

Figure Lengend Snippet: Effects of interfering PAR1, PAR3, or PAR4 expression on the thrombin-induced MIF production of astrocytes. a Western blot analysis of MIF protein levels in the astrocytes treated by 0–5 μM PAR1 inhibitor SCH79797 for 24 h in the presence of 100 nM thrombin. b Quantification data as shown in a . Quantities were normalized to endogenous β-actin. c MTT assay of SCH79797 effects on the cell viability of the astrocytes. d Interference efficiency of siRNA oligonucleotide for PAR3 was measured by RT-PCR, and siRNA1 was used for the knockdown experiments. e Western blot analysis of MIF in the astrocytes following PAR3 knockdown for 24 h, prior to stimulation with 100 nM thrombin for 24 h. Scrambles were used as control. f Quantification data as shown in e . Quantities were normalized to endogenous β-actin. (g) Western blot analysis of MIF protein levels in the astrocytes treated by 0–100 μM PAR4 inhibitor tcy-NH 2 for 24 h in the presence of 100 nM thrombin. h Quantification data as shown in g . Quantities were normalized to endogenous β-actin. i MTT assay of tcy-NH 2 effects on the cell viability of the astrocytes. Experiments were performed in triplicates. Error bars represent the SEM (* P < 0.05)

Article Snippet: For drug delivery, a total of 4.5 μl of 5 mM PAR1 inhibitor SCH79797 (R&D systems) was slowly injected intrathecally, prior to the incision suture.

Techniques: Expressing, Western Blot, MTT Assay, Reverse Transcription Polymerase Chain Reaction

Journal: Journal of Neuroinflammation

Article Title: Thrombin acts as inducer of proinflammatory macrophage migration inhibitory factor in astrocytes following rat spinal cord injury

doi: 10.1186/s12974-022-02488-w

Figure Lengend Snippet: Determination of the phosphorylated activation of the MAPKs/NFκB signals in the astrocytes following stimulation with thrombin. a Western blot analysis of phosphorylation of ERK, P38, JNK kinase, and p65NFκB protein after astrocyte stimulation with 0–200 nM thrombin for 24 h. b–e Quantification data as shown in a . Quantities were normalized to endogenous β-actin. f Western blot analysis of phosphorylation of ERK, P38, JNK kinase, and p65NFκB protein after astrocyte treatment with 0–5 μM SCH79797 for 24 h in the presence of 100 nM thrombin. g–j Quantification data as shown in f . Quantities were normalized to endogenous β-actin. Experiments were performed in triplicates. Error bars represent the SEM (* P < 0.05)

Article Snippet: For drug delivery, a total of 4.5 μl of 5 mM PAR1 inhibitor SCH79797 (R&D systems) was slowly injected intrathecally, prior to the incision suture.

Techniques: Activation Assay, Western Blot

Journal: Journal of Neuroinflammation

Article Title: Thrombin acts as inducer of proinflammatory macrophage migration inhibitory factor in astrocytes following rat spinal cord injury

doi: 10.1186/s12974-022-02488-w

Figure Lengend Snippet: Effects of PAR1 inhibition on the production of MIF following rat SCI. a Western blot analysis of MIF from 1 cm cord segments following injection of 4.5 μl of 5 mM PAR1 inhibitor SCH79797 or vehicle at lesion sites of the contused cord at 0, 1, 4, and 7d, respectively. b Quantification data as shown in a . Quantities were normalized to endogenous β-actin. Experiments were performed in triplicates. Error bars represent the SEM, * P < 0.05, # P < 0.05, two-way ANOVA with Tukey’s test. c–h Immunostaining for MIF expressed in S100β-positive astrocytes at 4d following SCH79797 treatment on the injured cord. Arrowheads indicate the positive signals. Scale bars, 500 μm in c , e , and g ; 50 μm in d , f , and h

Article Snippet: For drug delivery, a total of 4.5 μl of 5 mM PAR1 inhibitor SCH79797 (R&D systems) was slowly injected intrathecally, prior to the incision suture.

Techniques: Inhibition, Western Blot, Injection, Immunostaining

Journal: Journal of Neuroinflammation

Article Title: Thrombin acts as inducer of proinflammatory macrophage migration inhibitory factor in astrocytes following rat spinal cord injury

doi: 10.1186/s12974-022-02488-w

Figure Lengend Snippet: Effects of PAR1 inhibition on the recovery of motor function following rat SCI. a HE staining of the injured spinal cord at 21 d after injection of 4.5 μl of 5 mM PAR1 inhibitor SCH79797 or vehicle at lesion sites. b Quantification data as shown in a from eight animals each 3 sections. Lesion area was analyzed within 2000 μm either side of the lesion center based on the HE-negative proportion. c BBB score of hindlimbs analyzed two-way repeated measures ANOVA followed by Sidak's post hoc test at 0d, 7d, 14d, and 21d following intrathecal injection of 4.5 μl of 5 mM PAR1 inhibitor SCH79797 or vehicle at lesion sites. Error bars represent the SEM (* P < 0.05). Scale bars 1000 μm

Article Snippet: For drug delivery, a total of 4.5 μl of 5 mM PAR1 inhibitor SCH79797 (R&D systems) was slowly injected intrathecally, prior to the incision suture.

Techniques: Inhibition, Staining, Injection

Journal: Frontiers in Immunology

Article Title: Dabigatran and Wet AMD, Results From Retinal Pigment Epithelial Cell Monolayers, the Mouse Model of Choroidal Neovascularization, and Patients From the Medicare Data Base

doi: 10.3389/fimmu.2022.896274

Figure Lengend Snippet: Thrombin-mediated effects on CTGF and VEGF expression. (A) Cell lysates were analyzed for thrombin-mediated CTGF expression. Thrombin induced expression of CTGF, that was already maximal at 0.5 U/mL. (B) Densitometric analysis demonstrated that thrombin-induced CTGF expression could be reduced by dabigatran (10 μM), the protease activated receptor-1 (PAR1) receptor antagonist (SCH79797; 250 μM), and the non-specific protease inhibitor (α1-antitrypsin; 1 mg/mL), and could be triggered using PAR1-AP (30 μM), a PAR1 agonist. (C) VEGF secretion (assessed by ELISA) into the apical supernatant could be induced by thrombin treatment and reduced by dabigatran and the selective non-peptide PAR1 receptor antagonist SCH79797. VEGF secretion into the apical supernatant could be triggered using the PAR1-AP. Data are plotted as mean ± SEM; n = 3 independent experiments.

Article Snippet: Some of the cells were treated with thrombin (EMD Millipore Corp), complement components C3a, C5a (Complement Technology), PAR1 agonist PAR1-AP (Sigma-Aldrich; amino acid sequence SFLLRN), thrombin inhibitor dabigatran etexilate (Sigma-Aldrich), C3 inhibitor compstatin (R&D Systems), AP inhibitor TT30 (generously provided by Alexion Therapeutics), a protease inhibitor alpha1-antitrypsin (Sigma-Aldrich), and the thrombin receptor proteinase activated receptor 1 inhibitor SCH79797 (Sigma-Aldrich).

Techniques: Expressing, Protease Inhibitor, Enzyme-linked Immunosorbent Assay

Journal: Stem Cell Research & Therapy

Article Title: Thrombin promotes fibronectin secretion by bone marrow mesenchymal stem cells via the protease-activated receptor mediated signalling pathways

doi: 10.1186/scrt424

Figure Lengend Snippet: Primer sequences for PCR analyses

Article Snippet: Graded concentrations of thrombin were added and MSCs were incubated for 24 h, 48 h and 72 h. Also, the cells were exposed to small molecules, including the PAR1 antagonist (SCH79797, 1 μM, Santa Cruz Biotechnology, Santa Cruz, CA, USA), the PAR2 peptide antagonist (FSLLRY-NH 2 , 10 μM, Tocis Bioscience, Bristol, UK), the ERK1/2 inhibitor (PD98059, 20 μM, Sigma-Aldrich, Saint Louis, MO, USA), or the NFκB p65 inhibitor (ethyl pyruvate, 5 mM, Sigma-Aldrich, Saint Louis, MO, USA), for 30 minutes before the cells were treated with thrombin (4 U/ml).

Techniques: Sequencing