Journal: Wellcome Open Research

Article Title: Testing the effect of PAR1 inhibitors on Plasmodium falciparum -induced loss of endothelial cell barrier function

doi: 10.12688/wellcomeopenres.15602.3

Figure Lengend Snippet: Under normal circumstances, the presence of endothelial protein C receptor (EPCR)-activated protein C (aPC) on the endothelial surface is able to modulate the protease-activated receptor 1 (PAR1) response (by cleavage of R46) to thrombin along a cytoprotective pathway. In the presence of IE, the binding site for aPC can be occupied by P. falciparum erythrocyte membrane protein 1 (PfEMP1) or EPCR expression can be reduced, both limiting the availability of EPCR-aPC and resulting in cytopathic responses due to unmodified thrombin cleavage (R41) of PAR1. P. falciparum -infected erythrocytes (IE) are also able to release soluble factors that can influence endothelial integrity directly.

Article Snippet: In addition, we used the clinically licenced PAR1 inhibitor Vorapaxar (brand name Zontivity (Aralez Pharmaceuticals)), obtained as a non-clinical compound from Sigma-Aldrich.

Techniques: Binding Assay, Membrane, Expressing, Infection

Journal: Wellcome Open Research

Article Title: Testing the effect of PAR1 inhibitors on Plasmodium falciparum -induced loss of endothelial cell barrier function

doi: 10.12688/wellcomeopenres.15602.3

Figure Lengend Snippet: ( A ) Experimental timeline and cell index traces for P. falciparum infected erythrocyte (IE) lysate induced decrease in barrier function in HBMEC and the effect of the four inhibitors (0.3 µM). Schematic of the experimental timeline (not to scale) indicating the addition of IE lysate in the absence and presence of inhibitor to the cells. Cell index was normalised at the time point immediately prior to the addition of IE lysate, indicated by the black triangle in the timeline and cell index trace, and medium (black line) was set as baseline. The normalised cell index was determined at 2 and 16 hours after adding the lysate in the absence of inhibitor (green) or in the presence of Vorapaxar (blue), E5555 (magenta), #04 (cyan) or #91 (coral). ( B ) Graph of the data depicted in A. The decrease in normalised cell index between 2 and 16 hours was calculated and this ∆normalised cell index in absence of inhibitor was set at 100%. Shown is the mean ± SD of 5 independent experiments with * indicating a P-value <0.05 compared to no inhibitor.

Article Snippet: In addition, we used the clinically licenced PAR1 inhibitor Vorapaxar (brand name Zontivity (Aralez Pharmaceuticals)), obtained as a non-clinical compound from Sigma-Aldrich.

Techniques: Infection

Journal: Wellcome Open Research

Article Title: Testing the effect of PAR1 inhibitors on Plasmodium falciparum -induced loss of endothelial cell barrier function

doi: 10.12688/wellcomeopenres.15602.3

Figure Lengend Snippet: ( A ) Experimental timeline and representative cell index traces for P. falciparum infected erythrocyte (IE) lysate induced decrease in barrier function in HBMEC and the effect of 0.3 µM Vorapaxar. Schematic of the experimental timeline (not to scale) indicating the addition of IE lysate and Varopaxar. Cell index traces are shown for IE lysate in the absence (green) and presence of Varopaxar (blue) and medium in the absence (red) and presence of Varopaxar (cyan). Cell index was normalised at the time point immediately prior to the addition of IE lysate, indicated by the black triangle in the timeline and cell index trace, and medium (black line) was set as baseline. After 19 hours 2 nM thrombin was added as indicated by the arrow. ( B ) The same cell index traces as in A , but normalised at the time point prior to the addition of thrombin (black triangle). Vorapaxar (blue and cyan) reverses the effect of thrombin. ( C ) Graph of the inhibitor data analysed as depicted in B . The maximum decrease in normalised cell index by thrombin was determined for medium or lysate in the absence of inhibitor and set at 100%. Shown are the mean ± SD of 2 independent experiments, with * indicating a P-value <0.05 compared to no inhibitor.

Article Snippet: In addition, we used the clinically licenced PAR1 inhibitor Vorapaxar (brand name Zontivity (Aralez Pharmaceuticals)), obtained as a non-clinical compound from Sigma-Aldrich.

Techniques: Infection

Journal: CytoJournal

Article Title: A disintegrin-like and metalloproteinase 15 facilitates glioblastoma proliferation and metastasis through activation of the protease-activated receptor 1

doi: 10.25259/Cytojournal_92_2024

Figure Lengend Snippet: Inhibition of ADAM15-induced proliferation, migration, and invasion by PAR-1 antagonist treatment. (a) Evaluation by scratch assay after PAR-1 antagonist SCH79797 or PAR-2 antagonist FSLLRY-NH2 treatment. Scale bar = 100 μm. Objective: 100×. (b) Relative migration rate for scratch assay after PAR-1 antagonist SCH79797 or PAR-2 antagonist FSLLRY-NH2 treatment. (c) Evaluation by transwell assay with Matrigel invasion for 72 h. Scale bar = 50 μm. Objective: 200×. (d) Relative invasion rate for transwell assay after PAR-1 antagonist SCH79797 or PAR-2 antagonist FSLLRY-NH2 treatment. (e) Clonogenic assay in U251 and U87. (f) Measurement of colony number after PAR-1 antagonist SCH79797 or PAR-2 antagonist FSLLRY-NH2 treatment. n = 5 independent replicates. ns: No significant, ✶ ✶ ✶ P < 0.001. ADAM15: A disintegrin-like and metalloproteinase 15, PAR1: Protease-activated receptor 1.

Article Snippet: PAR1 antagonist: SCH79797 (Cat. No: SML1939, Merck Millipore, Billerica, MA, USA).

Techniques: Inhibition, Migration, Wound Healing Assay, Transwell Assay, Clonogenic Assay

Journal: CytoJournal

Article Title: A disintegrin-like and metalloproteinase 15 facilitates glioblastoma proliferation and metastasis through activation of the protease-activated receptor 1

doi: 10.25259/Cytojournal_92_2024

Figure Lengend Snippet: Inhibition of ADAM15-induced EMT by PAR-1 antagonist treatment. (a) Western blotting of E-cadherin, N-cadherin, and Vimentin protein expression after PAR-1 antagonist SCH79797 or PAR-2 antagonist FSLLRY-NH2 treatment in U251. (b) Histogram of E-cadherin, N-cadherin, and Vimentin protein expression in U251. (c) Western blotting of E-cadherin, N-cadherin, and Vimentin protein expression after PAR-1 antagonist SCH79797 or PAR-2 antagonist FSLLRY-NH2 treatment in U87. (d) Histogram of E-cadherin, N-cadherin, and Vimentin protein expression in U87. (e) Immunofluorescence of E-cadherin and Vimentin staining in U251 and U87. Scale bar = 20 μm. Objective: 400×. n = 5 independent replicates. ns: Not significant, ✶ ✶ P < 0.01. ADAM15: A disintegrin-like and metalloproteinase 15, PAR1: Protease-activated receptor 1, Epithelial-mesenchymal transition.

Article Snippet: PAR1 antagonist: SCH79797 (Cat. No: SML1939, Merck Millipore, Billerica, MA, USA).

Techniques: Inhibition, Western Blot, Expressing, Immunofluorescence, Staining

Journal: Frontiers in Pharmacology

Article Title: TAK-442, a Direct Factor Xa Inhibitor, Inhibits Monocyte Chemoattractant Protein 1 Production in Endothelial Cells via Involvement of Protease-Activated Receptor 1

doi: 10.3389/fphar.2018.01431

Figure Lengend Snippet: FXa- and thrombin-induced MCP-1 production in HUVECs. MCP-1 concentration was determined in cell supernatant collected 20 h after the addition of the agonist, FXa (A) or thrombin (B) . In inhibition studies (C,D) , each inhibitor was added 1 h prior to the addition of the agonist and effects of TAK-442 (TAK), melagatran (Mel), and vorapaxar (Vor) on MCP-1 productions induced by 1 U/mL FXa (C) and 0.3 U/mL thrombin (D) were measured. Data are shown as mean ± SEM ( n = 3). ∗ P ≤ 0.025 compared with the control value of cells treated without FXa or thrombin (one-tailed Williams’ test) (A,B) . ∗ P ≤ 0.025 and † P ≤ 0.05 compared with the control value of cells treated with FXa or thrombin and no inhibitor (one-tailed Williams’ test and Student’s t -test, respectively, following ANOVA) (C,D) .

Article Snippet: The selective FXa inhibitor TAK-442, 1-(1-{(2S)-3-[(6-chloro–2-naphthyl) sulfonyl]-2-hydroxypropanoyl}piperidin-4-yl)tetra hydropyrimidin-2(1H)-one, the selective thrombin inhibitor melagatran, N-((1R)-2-{(2S)–2-[({4-[amino(imino)methyl]benzyl}amino)carbonyl]azetidin-1-yl}-1–cyclohexyl-2-oxoethyl) glycine, and the selective PAR1 antagonist vorapaxar, ethyl(9-{(E)–2-[5-(3-fluorophenyl)pyridin-2-yl]vinyl}-1-methyl-3-oxododecahydronaphtho[2,3-c]furan-6-yl)carbamate, were synthesized at Takeda Pharmaceutical Co., Ltd. (Osaka, Japan).

Techniques: Concentration Assay, Inhibition, Control, One-tailed Test

Journal: Frontiers in Pharmacology

Article Title: TAK-442, a Direct Factor Xa Inhibitor, Inhibits Monocyte Chemoattractant Protein 1 Production in Endothelial Cells via Involvement of Protease-Activated Receptor 1

doi: 10.3389/fphar.2018.01431

Figure Lengend Snippet: Effects of TAK-442, melagatran, and vorapaxar on the intracellular calcium ion concentration ([Ca 2+ ]i) induced by FXa, thrombin, and SFLLRN-NH2 in human PAR1-transfected Chinese hamster ovary (hPAR1/CHO-K1) cells. Calcium signal was recorded after the addition of FXa (0.03 U/mL) (A) , thrombin (0.003 U/mL) (B) , or PAR1 agonist peptide SFLLRN-NH2 (3 nM) (C) using FRIPR. Each inhibitor, TAK-442, melagatran, or vorapaxar, was pre-incubated with the cells for 10 min before the treatment with each agonist. Data are expressed as the percentage inhibition of calcium signal obtained after the addition of agonist in inhibitor-treated wells ( n = 4) compared with control wells (no inhibitor added). The drug concentration need to suppress the [Ca 2+ ]i by 50% (IC 50 ) was determined.

Article Snippet: The selective FXa inhibitor TAK-442, 1-(1-{(2S)-3-[(6-chloro–2-naphthyl) sulfonyl]-2-hydroxypropanoyl}piperidin-4-yl)tetra hydropyrimidin-2(1H)-one, the selective thrombin inhibitor melagatran, N-((1R)-2-{(2S)–2-[({4-[amino(imino)methyl]benzyl}amino)carbonyl]azetidin-1-yl}-1–cyclohexyl-2-oxoethyl) glycine, and the selective PAR1 antagonist vorapaxar, ethyl(9-{(E)–2-[5-(3-fluorophenyl)pyridin-2-yl]vinyl}-1-methyl-3-oxododecahydronaphtho[2,3-c]furan-6-yl)carbamate, were synthesized at Takeda Pharmaceutical Co., Ltd. (Osaka, Japan).

Techniques: Concentration Assay, Transfection, Incubation, Inhibition, Control

Journal: Frontiers in Neuroscience

Article Title: Activity of Protease-Activated Receptors in Primary Cultured Human Myenteric Neurons

doi: 10.3389/fnins.2012.00133

Figure Lengend Snippet: PAR-APs and thrombin activate cultured myenteric neurons . Representative traces of voltage sensitive dye recordings showing neuronal responses to a 2 s spritz application (indicated by the horizontal gray bar) of PAR1-AP, PAR2-AP, PAR4-AP, and thrombin. Recordings were made in four 2 s long recording periods with 5–6 s intervals in between (indicated by the symbol between the traces). Every peak represents an action potential. (A) Representative traces from cultured human myenteric neurons to human specific PAR-APs and thrombin show comparable responses to PAR1-AP and thrombin but no response to PAR2-AP and a minor response to PAR4-AP. (B) Guinea pig cultured myenteric neurons fire action potentials in response to PAR1-AP, PAR2-AP, PAR4-AP, and thrombin; the PAR4 response is rather moderate.

Article Snippet: The PAR1 antagonist SCH79797 (N 3 -cyclopropyl-7-[[4-(1-methylethyl)phenyl] methyl]-7H-pyrrolo[3,2-f]quinazoline-1,3-diamine dihydrochloride; Biozol Diagnostica, Eching, Germany) was dissolved in waterfree 100% dimethyl sulfoxide (Acros Organics, Geel, Belgium; Ahn et al., ).

Techniques: Cell Culture

Journal: Frontiers in Neuroscience

Article Title: Activity of Protease-Activated Receptors in Primary Cultured Human Myenteric Neurons

doi: 10.3389/fnins.2012.00133

Figure Lengend Snippet: Analysis of neural actions of PAR-APs and thrombin in cultured human and guinea pig myenteric neurons . The graphs represent (from top to bottom) the proportion of neurons per cluster responding to a specific PAR activator, the specific PAR activator evoked spike frequency and the neuroindex which is the product of spike frequency and proportion of responding neurons. Data are illustrated with scatter plots showing the 25%/75% and the bars indicating the 10%/90% percentiles. PAR1-AP induced the strongest effect in human myenteric neurons, whereas PAR4-AP evoked only weak responses and PAR2-AP no response. Thrombin also activated human myenteric neurons but to a lesser degree than PAR1-AP. The PAR1-AP effect in human neurons is blocked by the specific PAR1 antagonist SCH79797. In guinea pig myenteric neurons the responsiveness to PAR1-AP, PAR2-AP, and thrombin was similar, but PAR4-AP evoked a spike discharge in less neurons at a significantly lower frequency. *Indicates significant differences to PAR1-AP in human neurons; # indicates significant differences to PAR1-AP in guinea pig neurons. Numbers in parenthesis indicate numbers of tissue/clusters/neurons.

Article Snippet: The PAR1 antagonist SCH79797 (N 3 -cyclopropyl-7-[[4-(1-methylethyl)phenyl] methyl]-7H-pyrrolo[3,2-f]quinazoline-1,3-diamine dihydrochloride; Biozol Diagnostica, Eching, Germany) was dissolved in waterfree 100% dimethyl sulfoxide (Acros Organics, Geel, Belgium; Ahn et al., ).

Techniques: Cell Culture

Journal: Frontiers in Neuroscience

Article Title: Activity of Protease-Activated Receptors in Primary Cultured Human Myenteric Neurons

doi: 10.3389/fnins.2012.00133

Figure Lengend Snippet: Response pattern to PAR-APs in guinea pig cultured myenteric neurons demonstrates receptor clustering . (A) Image shows outlines of a cluster of guinea pig cultured myenteric neurons stained with the voltage sensitive dye Di-8-ANEPPS. (B) Representative traces of a neuron [marked with a star in (A) ] that responded to PAR1-AP and PAR2-AP (2 s spritz application indicated by a gray horizontal bar). Recordings were made in four 2 s long recording periods with 5–6 s intervals in between (indicated by the symbol between the traces). Every peak represents an action potential. (C) Pairwise application of PAR-APs revealed functional coexpression patterns. Proportions of neurons responding to two PAR-APs are expressed relative to the proportion of neurons responding to any PAR-AP in that particular set of experiment (100%).

Article Snippet: The PAR1 antagonist SCH79797 (N 3 -cyclopropyl-7-[[4-(1-methylethyl)phenyl] methyl]-7H-pyrrolo[3,2-f]quinazoline-1,3-diamine dihydrochloride; Biozol Diagnostica, Eching, Germany) was dissolved in waterfree 100% dimethyl sulfoxide (Acros Organics, Geel, Belgium; Ahn et al., ).

Techniques: Cell Culture, Staining, Functional Assay