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14 protocols using Vorapaxar

PAR1-AP (TFLLR-NH2) and PAR4-AP (AYPGKF-NH2) were synthesized from Cosmogenetech (Seoul, Korea). Vorapaxar was purchased from Axon Medchem (Groningen, Netherlands), AZ3451 from Tocris Bio-techne (Minneapolis, MN, USA), BMS-986120 from Cayman Chemical (Ann Arbor, MI, USA), and other chemicals, unless otherwise indicated, were purchased from Sigma-Aldrich (St. Louis, MO, USA). The kinase inhibitor library used in HCS was purchased from Targetmol (Wellesley Hills, MA, USA).
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Serum-starved cells were preincubated at 37°C with 100 nM PF-543 (Tocris, #5754) or 10 µM W146 (Tocris, #3602) for 30 min, or with 1 µM MK-2206 (Selleck, #S1078) or 10 µM Vorapaxar (Axon Medchem, #1755) for 1 h.
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Bone marrow derived-human MSCs (Texas A&M) were incubated with vorapaxar (Axon Medchem, catalog #1755) 0.3 µM for 30 min to block PAR1, and then cells were washed, harvested, and administered in vivo to mice using the pneumonia model outlined above. Mouse survival was measured to determine the effect of PAR1 blockade on the therapeutic value of human MSCs.
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Reagents were obtained from the following suppliers: Evans Blue dye, TFLLR-NH2, bovine plasma thrombin, tetracycline hydrochloride, poly-L-lysine, and GSK1016790A were purchased from Sigma-Aldrich (Castle Hill, Australia); Fura2-AM was purchased from (ThermoFisher Scientific, Scoresby, Victoria). Vorapaxar (SCH-530348) was purchased from Axon Medchem (Groningen, The Netherlands) and HC067047 was purchased from Tocris Bioscience (Bristol, UK).
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Vorapaxar (SCH 530348) was obtained from Axon Medchem (Groningen, Netherlands) and I-343 was synthesized and purified by Dr. Luigi Aurelio (61 (link)). Both drugs were dissolved in dimethylformamide (DMF) (10 mg/ml) and then PBS (0.5% v/v) prior to administration at the indicated dose.
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Washed platelets were incubated with various concentrations of the PAR1 inhibitor vorapaxar (Axon Medchem, Reston, VA), the PAR4 inhibitor BMS-986120 (Cayman Chemical, Ann Arbor, MI), the P2Y12 inhibitors Ticagrelor (Selleckchem, Houston, TX) or 2-methylthioadenosine 5’-monophosphate triethylammonium salt hydrate (MeSAMP) (Sigma Aldrich, St. Louis, MO), or the cyclooxygenase inhibitor aspirin (ASA) (Sigma-Aldrich, St. Louis, MO), and maximal aggregation or calcium mobilization measured.
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Human α-Thrombin was from Enzyme Research Laboratories (#HT1002a). The following antibodies were from Cell Signaling Technologies including p44/42 MAPK ERK1/2 (#9102), phospho-p44/42 MAPK ERK1/2 (#9106), p38 MAPK (#8690), phospho-p38 MAPK (#4511), α-E-catenin (#3236), and phospho-α-E (Ser652)-catenin antibody (#13061). Horseradish peroxidase-conjugated goat-anti rabbit (#1706515) and goat-anti mouse antibodies (#1706516) were from BioRad. Vorapaxar or SCH 530348 (#1755) was from Axon MedChem, SB203580 (#S8307) was from Sigma-Aldrich, and U0126 (#U-6770) was from LC Laboratories.
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The PAR-1 inhibitor vorapaxar (Axon Medchem, Reston, VA, USA), PAR-1 agonist SFLLRN-NH2 (American Peptide Company, Sunnyvale, CA, USA), PAR2 agonists 2f-LIGRL-NH2 (EMD Millipore, Billerica, MD, USA), SLIGKV-NH2 (Tocris, Minneapolis, MN, USA), AC55541 (Tocris), TPCK-treated bovine trypsin (10,000 Nα-benzoyl-l-arginine ethyl ester units/mg, Sigma, St Louis, MO, USA), and human thrombin (1000 NIH units/mg; Sigma) were resuspended following the manufacturer’s instructions. Tryptase purified from human mast cells (70 units/mg, Fitzgerald, Acton, MA, USA) mixed with 15 kDa heparin from porcine stomach (Sigma) in a 1:10 molar ratio of tryptase to heparin immediately after thawing,38 (link) and PAR2 inhibitor ENMD-1068 (50 μg/ml; Enzo Life Sciences, Farmingdale, NY, USA) were used as previously described.39 (link) Tosyl-l-lysyl-chloromethane hydrochloride (TLCK; Sigma), an irreversible inhibitor of trypsin and trypsin-like serine proteases, was used at 10 nM, and the PAR-1 inhibitor vorapaxar was used at 25 μg/ml, both in RPMI/2% BSA.
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The V5‐tag antibody conjugated with fluorescein isothiocyanate (FITC) (catalog no. R963‐25) was from Invitrogen (Carlsbad, CA, USA). The Phospho‐p44/42 mitogen‐activated protein kinase (MAPK) (extracellular signal‐regulated kinase [ERK] 1/2) (Thr202/Tyr204) (D13.14.4E) Rabbit monoclonal antibody (mAb; catalog no. 4370) and p44/42 MAPK (ERK1/2) (3A7) Mouse mAb (catalog no. 9107) were both purchased from Cell Signaling Technology, Inc (Danvers, MA, USA). Trypsin (catalog # V511A) was from Promega (Fitchburg, WI, USA). The PAR1 activation peptide, SFLLRN‐NH2 and TFLLRN‐NH2 were from Tocris Bioscience (Minneapolis, MN, USA). PAR4 activation peptide, AYPGKF‐NH2, was from GenScript (Piscataway, NJ, USA). C4a (catalog no. A106, Lot 16) was from Complement Technology (Tyler, TX, USA). Human α‐thrombin (catalog # HCT‐0020, specific activity >2989 U/mg) was from Hematological Technologies (Essex Junction, VT, USA). Vorapaxar (catalog no. 1755, batch 2) was from Axon Medchem (Reston, VA, USA). All other reagents were from Thermo Fisher Scientific (Pittsburgh, PA, USA) except where noted.
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HIV-1 IIIB Tat recombinant protein was obtained through the AIDS Reagent Program, Division of AIDS, NIAID, NIH (Catalog # 2222). To protect against oxidation, Tat recombinant protein was reconstituted in PBS containing 0.5 mM DTT (dithiothreitol). Tat was used at a final concentration of 100 ng/mL and the DTT-containing reconstitution buffer served as control in experiments involving Tat. To rule out endotoxin contamination, Tat was heat-inactivated at 95° C for 30 minutes (data not shown). The broad-spectrum MMP inhibitor GM6001 (Tocris, Catalog # 2983) was reconstituted in DMSO (dimethyl sulfoxide) and used at a final concentration of 10 µM. Human recombinant pro-MMP-13 (R&D Systems, Catalog # 511-MM-010) and human recombinant catalytic MMP-13 (cMMP-13; Enzo Life Sciences, Catalog # BML-SE246–0010) were both used at a final concentration of 20 nM. Dose-curve experiments revealed that 20 nM of cMMP-13 resulted in maximal CCL2 release (data not shown). Human recombinant catalytic MMP-3 (cMMP-3; Millipore Calbiochem, Catalog # 444217) was used at a final concentration of 4.5 nM. Mouse recombinant IL-1β (R&D Systems, Catalog # 401-ML-CF) was used at 100 ng/mL. PBS was used as control in experiments involving IL-1β, pro-MMP-13, catalytic MMP-3, and catalytic MMP-13. Vorapaxar (Axon Medchem, Catalog # 1755) was reconstituted in DMSO and used at a final concentration of 2 µM.
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