Trap 6

Manufactured by AnaSpec

Sourced in Belgium

TRAP-6 is a synthetic hexapeptide that mimics the C-terminal sequence of the thrombin receptor. It is commonly used in research to activate the thrombin receptor and study its associated signaling pathways.

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Lab products found in correlation

Vcam 1, by Santa Cruz Biotechnology (2 mentions) Icam 1, by Santa Cruz Biotechnology (2 mentions) Hrp linked anti rabbit and anti mouse igg, by Cell Signaling Technology (1 mentions) Thrombin, by Merck Group (1 mentions) Texas red conjugated phalloidin, by Thermo Fisher Scientific (1 mentions) Pmypt, by Cell Signaling Technology (1 mentions) P120 catenin, by Cell Signaling Technology (1 mentions) β actin, by Cell Signaling Technology (1 mentions) Phospho nf κb, by Cell Signaling Technology (1 mentions) Ve cadherin, by Santa Cruz Biotechnology (1 mentions) E selectin, by Santa Cruz Biotechnology (1 mentions) Hpaec, by Lonza (1 mentions) Acetylated tubulin, by Merck Group (1 mentions) β tubulin, by Merck Group (1 mentions) Stathmin, by BD (1 mentions) End binding protein 1 eb1, by BD (1 mentions) Ionomycin, by Santa Cruz Biotechnology (1 mentions) Adenosine diphosphate (adp), by Chrono-Log (1 mentions) Taqman gene expression master mix, by Thermo Fisher Scientific (1 mentions) Pac 1, by BD (1 mentions)

7 protocols using trap 6

Pulmonary Endothelial Cell Culture Protocol

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Human pulmonary artery endothelial cells (HPAECs) were obtained from Lonza (Allendale, NJ) and used at passages 5–8. All experiments were performed in EGM growth medium (Lonza) containing 2% fetal bovine serum (FBS) unless otherwise specified. Texas Red–conjugated phalloidin and Alexa Fluor 488–labeled secondary antibodies were purchased form Molecular Probes (Eugene, OR). TRAP6 was obtained from AnaSpec (San Jose, CA). 8CPT was obtained from Calbiochem (La Jolla, CA). GGTI-298 and thrombin were obtained from Millipore-Sigma. Antibodies to diphospho–myosin light chain (MLC), pan-MLC, MYPT, pMYPT, p120-catenin, phospho-NFκB, β-actin, and tubulin antibodies were obtained from Cell Signaling (Beverly, MA); Rap1, VE-cadherin, ICAM1, VCAM1, and E-selectin antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). HRP-linked anti-mouse and anti-rabbit IgG were obtained from Cell Signaling (Beverly, MA). Unless otherwise specified, all biochemical reagents were obtained from Sigma (St. Louis, MO).

Ke Y., Karki P., Zhang C., Li Y., Nguyen T., Birukov K.G, & Birukova A.A. (2019). Mechanosensitive Rap1 activation promotes barrier function of lung vascular endothelium under cyclic stretch. Molecular Biology of the Cell, 30(8), 959-974.

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Platelet Aggregation Quantification

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Platelet aggregation was measured utilizing a light-transmittance aggregometer (PAP-8E; BioData Corporation). Following incubation with purge solution, PRP samples (100,000 platelets/µL) were re-calcified (1 mM CaCl₂) and stimulated to aggregate with the addition of 4 separate agonists: 32 µM thrombin receptor activating peptide 6 (TRAP-6; AnaSpec); 12 µM calcium ionophore (A23187); 20 µM adenosine diphosphate (ADP); or 5 µg/mL collagen. Immediately following agonist addition, light transmittance was recorded over a 10-min period. Samples were maintained at 37 °C and stirred at approximately 1000 rpm during aggregation recording. Area under the generated curve was recorded as a measure of platelet aggregation.

Ammann K.R., Outridge C.E., Roka-Moiia Y., Muslmani S., Ding J., Italiano J.E., Tomat E., Corbett S, & Slepian M.J. (2023). Sodium bicarbonate as a local adjunctive agent for limiting platelet activation, aggregation, and adhesion within cardiovascular therapeutic devices. Journal of Thrombosis and Thrombolysis, 56(3), 398-410.

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Endothelial Cell Signaling Protocol

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Human pulmonary artery endothelial cells (HPAEC) were obtained from Lonza (East Rutherford, NJ) and used for experiments at passages 5–8. ANP and TRAP6 were purchased from Ana Spec (San Jose, CA). Reagents for immunofluorescence were purchased from Molecular Probes (Eugene, OR). HRP-linked anti-mouse and -rabbit IgG antibodies were obtained from Cell Signaling Inc. (Beverly, MA). Antibodies to phospho- myosin phosphatase targeting subunit1 (MYPT), GEF-H1, diphospho-myosin light chain (MLC), phospho-stathmin, and IκBα were from Cell Signaling Inc (Beverly, MA); stathmin and End-Binding protein-1 (EB1) were from BD Transduction Laboratories (San Diego, CA); ICAM-1 and VCAM-1 were from Santa Cruz Biotechnology (Santa Cruz, CA). Peptidoglycan G (PepG) polymer covers cell surface of S. aureus Gram positive bacteria. PepG of 99% purity isolated from S. aureus, cat-77140, was obtained from Fluka (Buchs, Switzerland). Unless specified, other biochemical reagents including β-Tubulin and acetylated tubulin antibodies were obtained from Sigma (St. Louis, MO).

Tian Y., Mambetsariev I., Sarich N., Meng F, & Birukova A.A. (2014). Role of microtubules in attenuation of PepG-induced vascular endothelial dysfunction by atrial natriuretic peptide. Biochimica et biophysica acta, 1852(1), 104-119.

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Platelet-Derived Microparticle Generation

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Blood was collected from 4 female healthy donors (age 26–33 years) as described above, and platelets were isolated as previously described [47 (link)]. In brief, citrated blood was centrifuged at 120xg for 20 min at 20°C to obtain platelet-rich plasma (PRP). Platelets were isolated from PRP by centrifugation at 3,000xg for 2 min at 20°C in the presence of prostacyclin (PGI₂, 1 μM, Sigma-Aldrich, Austria), followed by repeated washing in sterile filtered (0.1 μm) phosphate-buffered saline (PBS: 133 mM NaCl, 1.3 mM KCl, 2.5 mM Na₂HPO₄x2H₂O, 0.5 mM KH₂PO₄, pH = 7.4).
MP generation was induced by addition of thrombin receptor activating peptide-6 (TRAP–6, 20 μM, AnaSpec, Belgium) or ionomycin (40 μM, Santa Cruz Biotechnology, Inc., Germany) in the presence and absence of pPCI (300 nM) to isolated platelets (300,000/μl) in PBS. After 20 min of stimulation platelets were centrifuged at 3,000xg for 5 min to obtain MP containing platelet-free supernatant.

Einfinger K., Badrnya S., Furtmüller M., Handschuh D., Lindner H, & Geiger M. (2015). Phospholipid Binding Protein C Inhibitor (PCI) Is Present on Microparticles Generated In Vitro and In Vivo. PLoS ONE, 10(11), e0143137.

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Platelet Aggregation Assay with Agonists

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LSPs and CPPs were diluted to 250,000 PLT/µL with corresponding PLT-poor plasma (PPP). Samples were treated with thrombin receptor-activating peptide 6 (TRAP-6) (20 µmol/L) from AnaSpec (Fremont, CA), collagen (5 µg/mL) or adenosine diphosphate (ADP) (20 µmol/L) (Chrono-Log Corp., Havertown, PA). The change in light transmission was recorded for 20 min using Chrono-log 700 (Chrono-Log Corp.). The maximum aggregation response was expressed as the maximum percentage change in light transmittance from baseline (26 (link)).

Tegegn T.Z., De Paoli S.H., Orecna M., Elhelu O.K., Woodle S.A., Tarandovskiy I.D., Ovanesov M.V, & Simak J. (2016). Characterization of procoagulant extracellular vesicles and platelet membrane disintegration in DMSO-cryopreserved platelets. Journal of Extracellular Vesicles, 5, 10.3402/jev.v5.30422.

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IL-1β Measurement in Human Cells

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Human IL-1β enzyme-linked immunosorbent assay kit was from Aviva System Biology. Anti–IL-1β antibody (3A6) and anti-GAPDH antibody (14C10) were from Cell Signaling. Primers for IL-1β (HS01555410), Caspase-1 (HS00354836), TLR2 (HS02621280), TLR4 (HS00152939), MYD88 (HS01573837), and GAPDH (HS02786624) as well as TaqMan Gene Expression Master Mix and complementary DNA Reverse Transcription Kit were from Applied Biosystems. Custom-made pre–IL-1β primer was from Eurofins. FAM-FLICA was from Immunochem Technologies. PAC-1, anti–P-selectin (clone AK4), and anti–IL-1β (clone AS10) were from BD Bioscience. TRAP-6 was from Anaspec. CRP-XL was from Cambcol. All other reagents were from Sigma Aldrich.

Berger M., Maqua H., Lysaja K., Mause S.F., Hindle M.S., Naseem K., Dahl E., Speer T., Marx N, & Schütt K. (2023). Platelets from patients with chronic inflammation have a phenotype of chronic IL-1β release. Research and Practice in Thrombosis and Haemostasis, 8(1), 102261.

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Isolation and Characterization of Platelet-Derived Extracellular Vesicles

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Washed platelets (2 × 10 8 /ml) were activated with different agonists (100 ng/ml convulxin, Enzo Life Sciences), 50 μM TRAP-6 (AnaSpec Inc.), or 5 nM thrombin (Haematologic Technologies) for 30 min at 37 °C. Integrin α IIb β 3 ligand binding was blocked with 10 nM tirofiban (CAS 144494-65-5, Correvio Int.). Integrin dependent outsidein signalling pathways were blocked with either 10 μM cytochalasin D (CAS 22144-77-0, Sigma), 10 μM PTPN1 inhibitor (CAS 765317-72-4, Merck), 10 μM SYK inhibitor (CAS 622387-85-3, Cayman Chemicals), 10 μM calpain inhibitor I (CAS 110044-82-1, Cayman chemicals), 1 μM ROCK (GSK429286A, selleckchem) or 10 μM Gα 13 inhibitor (made inhouse according to Shen B. et al. [28] , see below). Platelets were preincubated for 5 min at 37 °C with indicated inhibitors prior to activation, as described. Activated platelets were spun down by centrifugation at 300g for 5 min, after which the EV-containing supernatant was filtered with PK50 MiniSart sterile 0.8 μm filters (Sartorius). The filtrate was centrifuged at 20,000g for 1 h at 4 °C to pellet the EVs [22, 23, 29] . Pellets were resuspended in platelet buffer pH 7.45, snap frozen into liquid nitrogen and stored at -80 °C.

Heinzmann A.C.A., Karel M.F.A., Coenen D.M., Vajen T., Meulendijks N.M.M., Nagy M., Suylen D.P.L., Cosemans J.M.E.M., Heemskerk J.W.M., Hackeng T.M, & Koenen R.R. (2020). Complementary roles of platelet α_IIbβ₃ integrin, phosphatidylserine exposure and cytoskeletal rearrangement in the release of extracellular vesicles. Atherosclerosis, 310.

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