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R&D Systems par1 inhibitor sch79797
Par1 Inhibitor Sch79797, supplied by R&D Systems, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/par1+inhibitor+sch79797/pm38042209-56-5-10?v=R%26D+Systems
Average 90 stars, based on 1 article reviews

par1 inhibitor sch79797 - by Bioz Stars, 2026-08

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Effect of PAR1 blockage on hippocampal neuronal survival in glutamate toxicity and APC/AP9 treatment. Cells were cultured in NBM+ during days 9–10. Excitotoxicity was reached by substitution of NBM+ with buffered saline solutions (HBSS) containing glutamate (100 µM) for 40 min at 37 °C. PAR1 blocker SCH79797 (50 nM) was added to HBSS 30 min prior to Glu; APC (10 nM) or AP9 (20 µM) were added to HBSS 15 min prior to Glu. After 24 h, NBM+ was removed and cell viability was detected with the MTT assay kit as described in . Values are expressed as the mean ± S.D. of triplicate cultures. *— p < 0.05 compared to control group, #— p < 0.05 compared to group with glutamate (contr—with control HBSS containing 145 mM NaCl; 5 mM KCl; 1.8 mM CaCl 2 ; 1.0 mM MgCl 2 ; 20 mM HEPES; 5 mM glucose (pH 7.4), SCH—SCH79797).

Journal: International Journal of Molecular Sciences

Article Title: Neuroprotective Effects of Noncanonical PAR1 Agonists on Cultured Neurons in Excitotoxicity

doi: 10.3390/ijms25021221

Figure Lengend Snippet: Effect of PAR1 blockage on hippocampal neuronal survival in glutamate toxicity and APC/AP9 treatment. Cells were cultured in NBM+ during days 9–10. Excitotoxicity was reached by substitution of NBM+ with buffered saline solutions (HBSS) containing glutamate (100 µM) for 40 min at 37 °C. PAR1 blocker SCH79797 (50 nM) was added to HBSS 30 min prior to Glu; APC (10 nM) or AP9 (20 µM) were added to HBSS 15 min prior to Glu. After 24 h, NBM+ was removed and cell viability was detected with the MTT assay kit as described in . Values are expressed as the mean ± S.D. of triplicate cultures. *— p < 0.05 compared to control group, #— p < 0.05 compared to group with glutamate (contr—with control HBSS containing 145 mM NaCl; 5 mM KCl; 1.8 mM CaCl 2 ; 1.0 mM MgCl 2 ; 20 mM HEPES; 5 mM glucose (pH 7.4), SCH—SCH79797).

Article Snippet: Human APC, NaCl, KCl, CaCl 2 , MgCl 2 , KH 2 PO 4 , HEPES, glucose, glutamate, NMDA, Ara C and PAR1 inhibitor SCH79797 were from Sigma-Aldrich (St. Louis, MO, USA).

Techniques: Cell Culture, Saline, MTT Assay

APC and AP9 restore the Glu- and NMDA-induced [Ca 2+ ]i dysregulation in hippocampal neurons. ( A ) Glutamate-induced [Ca 2+ ]i alterations in cultured neurons and ( B ) changes of the areas under curves measured with Ca-dependent fluorescence Fluo-4 (effects of glutamate and PAR1 agonists), ( C ) NMDA-induced [Ca 2+ ]i alterations in cultured neurons and ( D ) changes of the areas under curves measured with Ca-dependent fluorescence Fluo-4 (effects of NMDA and PAR1 agonists). For [Ca 2+ ]i measurement, the cells were loaded with high-affinity Ca 2+ indicator Fluo-4 in the form of the acetoxymethyl (AM) ester (1–2 μM Fluo-4, 40 min, 37 °C). Fluo-4 fluorescence was excited at 488 nm and monitored at 505–535 nm. All measurements were carried out at 27–29 °C in HBSS. Glu or NMDA were washed out by a nominally calcium-free solution containing 0.1 mM EGTA instead of CaCl 2 and 2 mM MgCl 2 . For the measurement of the maximal signal at the final part of the experiments, a Ca 2+ ionophore ionomycin (2 μM) was applied in the presence of 5 mM Ca 2+ to saturate the indicator with Ca 2+ . *— p < 0.05 APC pretreatment compared to glutamate, **— p < 0.05 AP9 pretreatment compared to glutamate, #— p < 0.05 AP9 pretreatment compared to NMDA.

Journal: International Journal of Molecular Sciences

Article Title: Neuroprotective Effects of Noncanonical PAR1 Agonists on Cultured Neurons in Excitotoxicity

doi: 10.3390/ijms25021221

Figure Lengend Snippet: APC and AP9 restore the Glu- and NMDA-induced [Ca 2+ ]i dysregulation in hippocampal neurons. ( A ) Glutamate-induced [Ca 2+ ]i alterations in cultured neurons and ( B ) changes of the areas under curves measured with Ca-dependent fluorescence Fluo-4 (effects of glutamate and PAR1 agonists), ( C ) NMDA-induced [Ca 2+ ]i alterations in cultured neurons and ( D ) changes of the areas under curves measured with Ca-dependent fluorescence Fluo-4 (effects of NMDA and PAR1 agonists). For [Ca 2+ ]i measurement, the cells were loaded with high-affinity Ca 2+ indicator Fluo-4 in the form of the acetoxymethyl (AM) ester (1–2 μM Fluo-4, 40 min, 37 °C). Fluo-4 fluorescence was excited at 488 nm and monitored at 505–535 nm. All measurements were carried out at 27–29 °C in HBSS. Glu or NMDA were washed out by a nominally calcium-free solution containing 0.1 mM EGTA instead of CaCl 2 and 2 mM MgCl 2 . For the measurement of the maximal signal at the final part of the experiments, a Ca 2+ ionophore ionomycin (2 μM) was applied in the presence of 5 mM Ca 2+ to saturate the indicator with Ca 2+ . *— p < 0.05 APC pretreatment compared to glutamate, **— p < 0.05 AP9 pretreatment compared to glutamate, #— p < 0.05 AP9 pretreatment compared to NMDA.

Article Snippet: Human APC, NaCl, KCl, CaCl 2 , MgCl 2 , KH 2 PO 4 , HEPES, glucose, glutamate, NMDA, Ara C and PAR1 inhibitor SCH79797 were from Sigma-Aldrich (St. Louis, MO, USA).

Techniques: Cell Culture, Fluorescence

Biased signaling at PAR1 induced by APC and nanopeptide NPNDKYEPF-NH2 (AP9) analogs of the PAR1 tethered ligand liberated by APC. Though the exact mechanism underlying the neuroprotective effects of AP9 has not been discovered yet, it might function similarly to APC, promoting cytoprotective signaling via β-arrestin-2 and dishevelled-2 (Dvl-2) scaffold. In the present study, it was shown that the protective effects of APC and AP9 peptide are realized through stabilization of Glu- and NMDA-induced dysregulation of intracellular calcium concentration.

Journal: International Journal of Molecular Sciences

Article Title: Neuroprotective Effects of Noncanonical PAR1 Agonists on Cultured Neurons in Excitotoxicity

doi: 10.3390/ijms25021221

Figure Lengend Snippet: Biased signaling at PAR1 induced by APC and nanopeptide NPNDKYEPF-NH2 (AP9) analogs of the PAR1 tethered ligand liberated by APC. Though the exact mechanism underlying the neuroprotective effects of AP9 has not been discovered yet, it might function similarly to APC, promoting cytoprotective signaling via β-arrestin-2 and dishevelled-2 (Dvl-2) scaffold. In the present study, it was shown that the protective effects of APC and AP9 peptide are realized through stabilization of Glu- and NMDA-induced dysregulation of intracellular calcium concentration.

Article Snippet: Human APC, NaCl, KCl, CaCl 2 , MgCl 2 , KH 2 PO 4 , HEPES, glucose, glutamate, NMDA, Ara C and PAR1 inhibitor SCH79797 were from Sigma-Aldrich (St. Louis, MO, USA).

Techniques: Concentration Assay

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

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

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

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

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)

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

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)

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

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

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

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

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

(A-C) Immunofluorescence of PAR1 (green) in myometrium from a pregnant woman (A) , non-pregnant woman ( B ), and fetal membrane (C) . Nuclei were stained with DAPI (blue). Am, amnion, Cho, chorion, Deci, decidua. (D-G) Localization of hemorrhage, thrombin, and PAR1 in placental abruption at 25 weeks of gestation (D and E) , and 33 weeks gestation (F and G) resulting in disseminated intravascular coagulopathy and uterine bleeding requiring hysterectomy for hemostasis. (D and F) Hematoxylin and eosin staining of the myometrium adjacent to the placenta. Note that hemorrhage infiltrated the myometrium. Bars, 50 μm. (E and G) Immunofluorescence of PAR1 (green), thrombin (red), and DAPI (blue) at the same location of (A) . Bars, 50 μm.

Journal: PLoS ONE

Article Title: Mechanisms of thrombin-Induced myometrial contractions: Potential targets of progesterone

doi: 10.1371/journal.pone.0231944

Figure Lengend Snippet: (A-C) Immunofluorescence of PAR1 (green) in myometrium from a pregnant woman (A) , non-pregnant woman ( B ), and fetal membrane (C) . Nuclei were stained with DAPI (blue). Am, amnion, Cho, chorion, Deci, decidua. (D-G) Localization of hemorrhage, thrombin, and PAR1 in placental abruption at 25 weeks of gestation (D and E) , and 33 weeks gestation (F and G) resulting in disseminated intravascular coagulopathy and uterine bleeding requiring hysterectomy for hemostasis. (D and F) Hematoxylin and eosin staining of the myometrium adjacent to the placenta. Note that hemorrhage infiltrated the myometrium. Bars, 50 μm. (E and G) Immunofluorescence of PAR1 (green), thrombin (red), and DAPI (blue) at the same location of (A) . Bars, 50 μm.

Article Snippet: On the day of experiments, cells in collagen gels were pretreated with the following reagents for 1 h: 100 nM PAR1 selective antagonist SCH79797 (No.1592 Tocris), 1 μM ROCK inhibitor Y-27632 (10005583, Cayman Chemical), 10 μM MLCK inhibitor, ML-7 (11801, Cayman Chemical), 10 μM of indomethacin (I-7378, Sigma), or 1 μM of progesterone (28921–64, Nacalai-tesque).

Techniques: Immunofluorescence, Membrane, Staining

Thrombin increased contraction of primary human myometrial cells through PAR1. (A and B) (Left images) Representative images of collagen lattice assay of human myometrial cells at 30 min treated with PBS (Ctl) and thrombin (A) or PAR1 activating peptide, TFLLR (B) . (Right graphs) Quantification of myometrial contractions in collagen lattice assays (n = 3). **, p < 0.01 at each time point. (C) Collagen lattice assay of myometrial cells at 30 min with 2 U/mL of thrombin (Thr) pretreated with or without 100 nM PAR1 selective inhibitor (SCH79797, PAR1-i) for 1 h (n = 3). Representative image (upper panel) and quantification of gel areas (lower graph). The experiments were repeated three times. *, p < 0.05, and **, p < 0.01.

Journal: PLoS ONE

Article Title: Mechanisms of thrombin-Induced myometrial contractions: Potential targets of progesterone

doi: 10.1371/journal.pone.0231944

Figure Lengend Snippet: Thrombin increased contraction of primary human myometrial cells through PAR1. (A and B) (Left images) Representative images of collagen lattice assay of human myometrial cells at 30 min treated with PBS (Ctl) and thrombin (A) or PAR1 activating peptide, TFLLR (B) . (Right graphs) Quantification of myometrial contractions in collagen lattice assays (n = 3). **, p < 0.01 at each time point. (C) Collagen lattice assay of myometrial cells at 30 min with 2 U/mL of thrombin (Thr) pretreated with or without 100 nM PAR1 selective inhibitor (SCH79797, PAR1-i) for 1 h (n = 3). Representative image (upper panel) and quantification of gel areas (lower graph). The experiments were repeated three times. *, p < 0.05, and **, p < 0.01.

Techniques:

(A) Immunocytochemistry of PAR1 (red) and phosphorylated MLC2 (Ser19, green) in human myometrial cells. (B, C) Immunoblots of phosphorylated MLC2 (p-MLC2), total MLC2, and β-actin of myometrial cells. Myometrial cells were treated with thrombin (2 U/mL) as a function of time (B) , or pretreated with 100 nM PAR1 inhibitor (SCH79797) for 1 h, and then treated with 2 U/mL of thrombin for 30 min (C) . The experiments were repeated three times.

Journal: PLoS ONE

Article Title: Mechanisms of thrombin-Induced myometrial contractions: Potential targets of progesterone

doi: 10.1371/journal.pone.0231944

Figure Lengend Snippet: (A) Immunocytochemistry of PAR1 (red) and phosphorylated MLC2 (Ser19, green) in human myometrial cells. (B, C) Immunoblots of phosphorylated MLC2 (p-MLC2), total MLC2, and β-actin of myometrial cells. Myometrial cells were treated with thrombin (2 U/mL) as a function of time (B) , or pretreated with 100 nM PAR1 inhibitor (SCH79797) for 1 h, and then treated with 2 U/mL of thrombin for 30 min (C) . The experiments were repeated three times.

Techniques: Immunocytochemistry, Western Blot

(A) Collagen lattice assay of myometrial cells at 30 min with 2 U/mL of thrombin (Thr) pretreated with 1 μM progesterone (P4) for 1 h. Representative image (upper panel) and quantification of gel areas (lower graph). (B) Inhibition of thrombin-induced increases of PTGS2 , IL1B , and F2R mRNA by P4. Myometrial cells were pretreated with 1 μM of P4 for 1 h, and then treated with 2 U/mL of thrombin. (C) Gene expressions of progesterone receptor-A and–B ( PgR-A and PgR-B ) with 24 h treatment of 1 U/mL of thrombin, 10 nM of PGE2, and 10 nM of PGF2α (upper graphs) and PgR-A to PgR-B ratio (lower graphs). n = 3 in each group. *, p < 0.05, and **, p < 0.01. The experiments were repeated three times.

Journal: PLoS ONE

Article Title: Mechanisms of thrombin-Induced myometrial contractions: Potential targets of progesterone

doi: 10.1371/journal.pone.0231944

Figure Lengend Snippet: (A) Collagen lattice assay of myometrial cells at 30 min with 2 U/mL of thrombin (Thr) pretreated with 1 μM progesterone (P4) for 1 h. Representative image (upper panel) and quantification of gel areas (lower graph). (B) Inhibition of thrombin-induced increases of PTGS2 , IL1B , and F2R mRNA by P4. Myometrial cells were pretreated with 1 μM of P4 for 1 h, and then treated with 2 U/mL of thrombin. (C) Gene expressions of progesterone receptor-A and–B ( PgR-A and PgR-B ) with 24 h treatment of 1 U/mL of thrombin, 10 nM of PGE2, and 10 nM of PGF2α (upper graphs) and PgR-A to PgR-B ratio (lower graphs). n = 3 in each group. *, p < 0.05, and **, p < 0.01. The experiments were repeated three times.

Techniques: Inhibition

Blood-gas-analysis and MAP: in both arms of the study (thrombin blockage with argatroban and PAR1-antagonism with SCH79797) two arterial blood samples were taken

Journal: BMC Neuroscience

Article Title: Thrombin contributes to the injury development and neurological deficit after acute subdural hemorrhage in rats only in collaboration with additional blood-derived factors

doi: 10.1186/s12868-018-0481-5

Figure Lengend Snippet: Blood-gas-analysis and MAP: in both arms of the study (thrombin blockage with argatroban and PAR1-antagonism with SCH79797) two arterial blood samples were taken

Article Snippet: Vehicle and treatment groups received a subdural infusion of 300 μl autologous blood in combination with an i.c.v. injection of either 10 μl vehicle (DMSO), or the PAR1 inhibitor SCH79797 (Axon Medchem, Groningen, NL).

Techniques:

The local CBF was not affected by the PAR1-antagonist SCH 79797 after ASDH (left graph). CBF was stable during the baseline period (32.6 ± 0.5 LDU, mean ± SEM). In the second minute after ASDH induction CBF dropped down and was constantly significant underneath the sham-operated animals (**SCH-1 vs. sham P < 0.001; **SCH-5 vs. sham P < 0.001; **vehicle vs. sham P < 0.001). Local administration of the PAR1 antagonist SCH79797 results in a not significantly reduced neuronal damage 14 days after ASDH compared to vehicle treatment, similar to the Argatroban treatment (right graph)

Journal: BMC Neuroscience

Article Title: Thrombin contributes to the injury development and neurological deficit after acute subdural hemorrhage in rats only in collaboration with additional blood-derived factors

doi: 10.1186/s12868-018-0481-5

Figure Lengend Snippet: The local CBF was not affected by the PAR1-antagonist SCH 79797 after ASDH (left graph). CBF was stable during the baseline period (32.6 ± 0.5 LDU, mean ± SEM). In the second minute after ASDH induction CBF dropped down and was constantly significant underneath the sham-operated animals (**SCH-1 vs. sham P < 0.001; **SCH-5 vs. sham P < 0.001; **vehicle vs. sham P < 0.001). Local administration of the PAR1 antagonist SCH79797 results in a not significantly reduced neuronal damage 14 days after ASDH compared to vehicle treatment, similar to the Argatroban treatment (right graph)

Techniques:

PAR1 is expressed by neural stem cells in the sub-ventricular zone (SVZ) of the adult mouse brain. Photomicrographs show immunofluorescence double-labeling for PAR1 with Sox2-positive ( A ), or PAR1 with Nestin-positive ( C ) multipotent neural stem cells (NSCs) within the lateral wall of the lateral ventricle (LV). RNAscope was used to identify cells expressing both PAR1 and Sox2 ( B ), or Nestin ( D ) RNA in NSCs of the adult SVZ. Arrow indicates an example of a double-labeled cell in each case, with arrowhead indicating a singly labeled cell (Scale bar = 10 μm). Boxed area in B and D is also shown at higher magnification to visualize double-labeled cells. ( E ) Histogram shows expression of PAR1 RNA was high in NSCs grown as neurospheres (NS), or when plated on poly-L-lysine coated coverslips as monolayers in stem cell media containing EGF and bFGF. PAR1 RNA expression by NSC monolayers decreased by 87% when EGF and bFGF were removed from the media for 7 DIV promoting stem cell differentiation. ( F ) Withdrawal of EGF and bFGF to induce NSC monolayer differentiation resulted in a parallel decrease in Nestin RNA expression. (**P < 0.01, ***P < 0.001 Students t-test).

Journal: Scientific Reports

Article Title: The Thrombin Receptor Restricts Subventricular Zone Neural Stem Cell Expansion and Differentiation

doi: 10.1038/s41598-018-27613-9

Figure Lengend Snippet: PAR1 is expressed by neural stem cells in the sub-ventricular zone (SVZ) of the adult mouse brain. Photomicrographs show immunofluorescence double-labeling for PAR1 with Sox2-positive ( A ), or PAR1 with Nestin-positive ( C ) multipotent neural stem cells (NSCs) within the lateral wall of the lateral ventricle (LV). RNAscope was used to identify cells expressing both PAR1 and Sox2 ( B ), or Nestin ( D ) RNA in NSCs of the adult SVZ. Arrow indicates an example of a double-labeled cell in each case, with arrowhead indicating a singly labeled cell (Scale bar = 10 μm). Boxed area in B and D is also shown at higher magnification to visualize double-labeled cells. ( E ) Histogram shows expression of PAR1 RNA was high in NSCs grown as neurospheres (NS), or when plated on poly-L-lysine coated coverslips as monolayers in stem cell media containing EGF and bFGF. PAR1 RNA expression by NSC monolayers decreased by 87% when EGF and bFGF were removed from the media for 7 DIV promoting stem cell differentiation. ( F ) Withdrawal of EGF and bFGF to induce NSC monolayer differentiation resulted in a parallel decrease in Nestin RNA expression. (**P < 0.01, ***P < 0.001 Students t-test).

Article Snippet: Alternatively, the effect of PAR1 inactivation was evaluated by application of a PAR1 specific small molecule inhibitor (SCH79797, 35 or 70 nM, Tocris, Bristol, United Kingdom) applied to PAR1+/+ NSCs.

Techniques: Immunofluorescence, Labeling, RNAscope, Expressing, RNA Expression, Cell Differentiation

Thrombin gates Ca 2+ signaling in neural stem cells in a PAR1-dependent manner. Traces, photomicrographs, and histograms ( A – J ), show that thrombin-induced Ca 2+ signaling in NSCs occurs in a manner that depends in part on the presence of PAR1. The change in fluorescence intensity measured in response to application of ( A ) the PAR1 agonist thrombin (5 μg/mL, (135 nM)), or ( C ) a PAR1-activating peptide (PAR1-AP, 40 μM) to Rhod-3 loaded NSCs monolayer cultures grown in the presence of EGF and bFGF. Subsequent application of ( A ) PAR1-AP or ( C ) thrombin 240 s later resulted in significantly lower ΔF/F0, indicating receptor desensitization by the first agonist. Application of thrombin ( E – G ), or PAR1-AP ( H – J ), to NSCs derived from PAR1+/+ or PAR1−/− mice demonstrates the absence of Ca 2+ signaling in response to either agonist in NSCs lacking PAR1. The change in intensity (ΔF) over baseline intensity (F0) is provided and expressed as ΔF/F0 = [(F − F0)/F0]. (***P < 0.001, Scale bar = 50 μm).

Journal: Scientific Reports

Article Title: The Thrombin Receptor Restricts Subventricular Zone Neural Stem Cell Expansion and Differentiation

doi: 10.1038/s41598-018-27613-9

Figure Lengend Snippet: Thrombin gates Ca 2+ signaling in neural stem cells in a PAR1-dependent manner. Traces, photomicrographs, and histograms ( A – J ), show that thrombin-induced Ca 2+ signaling in NSCs occurs in a manner that depends in part on the presence of PAR1. The change in fluorescence intensity measured in response to application of ( A ) the PAR1 agonist thrombin (5 μg/mL, (135 nM)), or ( C ) a PAR1-activating peptide (PAR1-AP, 40 μM) to Rhod-3 loaded NSCs monolayer cultures grown in the presence of EGF and bFGF. Subsequent application of ( A ) PAR1-AP or ( C ) thrombin 240 s later resulted in significantly lower ΔF/F0, indicating receptor desensitization by the first agonist. Application of thrombin ( E – G ), or PAR1-AP ( H – J ), to NSCs derived from PAR1+/+ or PAR1−/− mice demonstrates the absence of Ca 2+ signaling in response to either agonist in NSCs lacking PAR1. The change in intensity (ΔF) over baseline intensity (F0) is provided and expressed as ΔF/F0 = [(F − F0)/F0]. (***P < 0.001, Scale bar = 50 μm).

Techniques: Fluorescence, Derivative Assay

Blocking PAR1 promotes neural stem cell proliferation. Histograms and photomicrographs ( A – D ) show that NSCs lacking PAR1 have increased capacity to form neurosphere (NS) in culture and demonstrate higher levels of BrdU incorporation compared to those derived from PAR1+/+ mice (Scale bar = 100 μm). Treatment of NSCs with a PAR1 small molecule inhibitor, SCH79797 (35 or 70 nM) for 18 h increased BrdU incorporation ( E ), whereas a PAR1 activating peptide (PAR1-AP, 40 μM) reduced BrdU incorporation ( F ). (*P < 0.05, **P < 0.01, ***P < 0.001 Students t-test).

Journal: Scientific Reports

Article Title: The Thrombin Receptor Restricts Subventricular Zone Neural Stem Cell Expansion and Differentiation

doi: 10.1038/s41598-018-27613-9

Figure Lengend Snippet: Blocking PAR1 promotes neural stem cell proliferation. Histograms and photomicrographs ( A – D ) show that NSCs lacking PAR1 have increased capacity to form neurosphere (NS) in culture and demonstrate higher levels of BrdU incorporation compared to those derived from PAR1+/+ mice (Scale bar = 100 μm). Treatment of NSCs with a PAR1 small molecule inhibitor, SCH79797 (35 or 70 nM) for 18 h increased BrdU incorporation ( E ), whereas a PAR1 activating peptide (PAR1-AP, 40 μM) reduced BrdU incorporation ( F ). (*P < 0.05, **P < 0.01, ***P < 0.001 Students t-test).

Techniques: Blocking Assay, BrdU Incorporation Assay, Derivative Assay

PAR1 gene knockout enhances neural stem cell expansion and oligodendrocyte differentiation in cell culture. Photomicrographs and cell counts normalized to DAPI show significantly increased immunofluorescence for NG2, a marker for NSCs ( A – C ), Olig2 a marker of oligodendrocyte progenitor cells and young oligodendrocytes ( D – F ), and PLP, a marker for mature oligodendrocytes ( G – I ) in NSC cultures grown in the presence or absence of EGF and bFGF. The percentage of NG2, Olig2 and PLP+ cells was greater in NSC cultures lacking PAR1 when grown in the presence of EGF and bFGF (2 DIV). After withdrawal of EGF and bFGF for 5 DIV, we observed an expected decrease in the percentage of cells positive for NG2 and an increase in those positive for PLP. The increase in PLP+ cells after withdrawal of EGF and bFGF was greater in NSCs lacking PAR1 compared to wild type NSCs. (Scale bar = 50 μm). (*P < 0.05, **P < 0.01, ***P < 0.001, Students t-test).

Journal: Scientific Reports

Article Title: The Thrombin Receptor Restricts Subventricular Zone Neural Stem Cell Expansion and Differentiation

doi: 10.1038/s41598-018-27613-9

Figure Lengend Snippet: PAR1 gene knockout enhances neural stem cell expansion and oligodendrocyte differentiation in cell culture. Photomicrographs and cell counts normalized to DAPI show significantly increased immunofluorescence for NG2, a marker for NSCs ( A – C ), Olig2 a marker of oligodendrocyte progenitor cells and young oligodendrocytes ( D – F ), and PLP, a marker for mature oligodendrocytes ( G – I ) in NSC cultures grown in the presence or absence of EGF and bFGF. The percentage of NG2, Olig2 and PLP+ cells was greater in NSC cultures lacking PAR1 when grown in the presence of EGF and bFGF (2 DIV). After withdrawal of EGF and bFGF for 5 DIV, we observed an expected decrease in the percentage of cells positive for NG2 and an increase in those positive for PLP. The increase in PLP+ cells after withdrawal of EGF and bFGF was greater in NSCs lacking PAR1 compared to wild type NSCs. (Scale bar = 50 μm). (*P < 0.05, **P < 0.01, ***P < 0.001, Students t-test).

Techniques: Gene Knockout, Cell Culture, Immunofluorescence, Marker

SVZ NSCs with PAR1 gene knockout show enhanced differentiation in vitro . ( A ) Expression of RNA indicative of the neural stem cell stage of differentiation (Nestin) was greater in NSCs derived from the SVZ of adult PAR1−/− compared to PAR1+/+ mice when grown in the presence of EGF and bFGF. The rapid loss of Nestin RNA in NSCs by 3 and at 7 d after withdrawal of EGF and bFGF is consistent with their differentiation and did not differ across genotypes. ( B ) Olig2, a marker of oligodendrocyte progenitor cells and young oligodendrocytes was higher in PAR1−/− NSCs grown in the presence of EGF and bFGF and lower by 3 d after growth factor withdrawal. Expression of RNA encoding MBP ( C ) and PLP ( D ) were greatly increased by growth factor withdrawal and each was expressed at highest levels in NSCs lacking PAR1 at 3 d, with MBP remaining significantly higher after 7 d of differentiation. ( E ) Growth factor withdrawal also resulted in an increase in GFAP ( E ) and Neurofilament ( F ) expression, with differentiating NSCs lacking PAR1 showing higher levels of GFAP but lower levels of neurofilament. (*P < 0.05, **P < 0.01, ***P < 0.001 Students t-test).

Journal: Scientific Reports

Article Title: The Thrombin Receptor Restricts Subventricular Zone Neural Stem Cell Expansion and Differentiation

doi: 10.1038/s41598-018-27613-9

Figure Lengend Snippet: SVZ NSCs with PAR1 gene knockout show enhanced differentiation in vitro . ( A ) Expression of RNA indicative of the neural stem cell stage of differentiation (Nestin) was greater in NSCs derived from the SVZ of adult PAR1−/− compared to PAR1+/+ mice when grown in the presence of EGF and bFGF. The rapid loss of Nestin RNA in NSCs by 3 and at 7 d after withdrawal of EGF and bFGF is consistent with their differentiation and did not differ across genotypes. ( B ) Olig2, a marker of oligodendrocyte progenitor cells and young oligodendrocytes was higher in PAR1−/− NSCs grown in the presence of EGF and bFGF and lower by 3 d after growth factor withdrawal. Expression of RNA encoding MBP ( C ) and PLP ( D ) were greatly increased by growth factor withdrawal and each was expressed at highest levels in NSCs lacking PAR1 at 3 d, with MBP remaining significantly higher after 7 d of differentiation. ( E ) Growth factor withdrawal also resulted in an increase in GFAP ( E ) and Neurofilament ( F ) expression, with differentiating NSCs lacking PAR1 showing higher levels of GFAP but lower levels of neurofilament. (*P < 0.05, **P < 0.01, ***P < 0.001 Students t-test).

Techniques: Gene Knockout, In Vitro, Expressing, Derivative Assay, Marker

PAR1 gene knockout results in increased numbers of Sox2+ neural stem cells and oligodendrocyte progenitor cells in the adult SVZ. Counts of Sox2-positive NSCs ( C – E ), and Ki-67-positive proliferating cells ( F – H ) were greater in coronal sections taken +0.5 mm to Bregma ( A , B ) through the SVZ of PAR1−/− compared to PAR1+/+ adult mice. A greater number of cells positive for the early oligodendrocyte lineage marker Olig2 were also observed in PAR1−/− mice in parallel sections ( I – K ). Ki-67 and Olig2 stained sections were counterstained with methyl green. (*P < 0.05, **P < 0.01, Students t-test) (Scale bar = 50 μm).

Journal: Scientific Reports

Article Title: The Thrombin Receptor Restricts Subventricular Zone Neural Stem Cell Expansion and Differentiation

doi: 10.1038/s41598-018-27613-9

Figure Lengend Snippet: PAR1 gene knockout results in increased numbers of Sox2+ neural stem cells and oligodendrocyte progenitor cells in the adult SVZ. Counts of Sox2-positive NSCs ( C – E ), and Ki-67-positive proliferating cells ( F – H ) were greater in coronal sections taken +0.5 mm to Bregma ( A , B ) through the SVZ of PAR1−/− compared to PAR1+/+ adult mice. A greater number of cells positive for the early oligodendrocyte lineage marker Olig2 were also observed in PAR1−/− mice in parallel sections ( I – K ). Ki-67 and Olig2 stained sections were counterstained with methyl green. (*P < 0.05, **P < 0.01, Students t-test) (Scale bar = 50 μm).

Techniques: Gene Knockout, Marker, Staining

PAR1 gene knockout results in increased numbers of oligodendrocytes in the corpus callosum and anterior commissure of the adult mouse brain. The corpus callosum of adult (8 wk) PAR1−/− mice showed an increase in the number of Olig2- ( A – C ) and CC-1-positive cells ( D – F ) relative to PAR1+/+ mice. Similar increases in Olig2 ( J – L ) and CC-1 ( M – O ) were also observed in the anterior commissure of mice with PAR1 gene knockout. Counts of GFAP+ astrocytes in parallel sections did not differ between genotypes ( G – I and P – R ). All data are expressed as the mean number of positive cells counted per area ± SEM. (*P < 0.05, **P < 0.01, ***P < 0.001, Students t-test) (Scale bar = 50 μm).

Journal: Scientific Reports

Article Title: The Thrombin Receptor Restricts Subventricular Zone Neural Stem Cell Expansion and Differentiation

doi: 10.1038/s41598-018-27613-9

Figure Lengend Snippet: PAR1 gene knockout results in increased numbers of oligodendrocytes in the corpus callosum and anterior commissure of the adult mouse brain. The corpus callosum of adult (8 wk) PAR1−/− mice showed an increase in the number of Olig2- ( A – C ) and CC-1-positive cells ( D – F ) relative to PAR1+/+ mice. Similar increases in Olig2 ( J – L ) and CC-1 ( M – O ) were also observed in the anterior commissure of mice with PAR1 gene knockout. Counts of GFAP+ astrocytes in parallel sections did not differ between genotypes ( G – I and P – R ). All data are expressed as the mean number of positive cells counted per area ± SEM. (*P < 0.05, **P < 0.01, ***P < 0.001, Students t-test) (Scale bar = 50 μm).

Techniques: Gene Knockout

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