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Ar c66096

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AR-C66096 is a small molecule inhibitor that targets the Interleukin-1 receptor (IL-1R). It is designed for use in research applications.

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Lab products found in correlation

Fluo 4 am, by Thermo Fisher Scientific (2 mentions) Lps from e coli 0111 b4, by Merck Group (2 mentions) 3 isobutyl 1 methyl xanthine (ibmx), by Bio-Techne (2 mentions) Rolipram, by Bio-Techne (2 mentions) Adenosine diphosphate (adp), by Bio-Techne (2 mentions) Ar c 69931, by Bio-Techne (2 mentions) Psb0739, by Bio-Techne (1 mentions) Thrombin, by Merck Group (1 mentions) Anti akt, by Cell Signaling Technology (1 mentions) Mrs2395, by Merck Group (1 mentions) Rottlerin, by Bio-Techne (1 mentions) Trap 6, by Bio-Techne (1 mentions) Pac 1 fitc, by BD (1 mentions) Phospho akt ser473, by Cell Signaling Technology (1 mentions) U73343, by Bio-Techne (1 mentions) U73122, by Bio-Techne (1 mentions) P gsk3β ser9, by Cell Signaling Technology (1 mentions) Go6976, by Bio-Techne (1 mentions) Mrs2179, by Bio-Techne (1 mentions) Anti cd62p apc, by BD (1 mentions)

Spelling variants of this product

AR‐C66096 tetrasodium salt (2 citations)

7 protocols using ar c66096

1

Platelet activation signaling pathways

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MRS2395 [2,2-Dimethyl-propionic acid 3-(2-chloro-6-methylaminopurin-9-yl)-2-(2,2-dimethyl-propionyloxymethyl)-propyl
ester], adenosine diphosphate (ADP), wortmannin, thrombin and all other chemicals and reagents were purchased from Sigma-Aldrich
(St Louis, MO, USA) or previously mentioned sources unless specified otherwise.[12 (link), 13 (link)] TRAP-6 (SFLLRN), U73122, U73343, Ro 31–8220, Rottlerin, Go6976, MRS2179, BAPTA,
PSB 0739 and AR-C 66096 were obtained from Tocris (Bristol, UK). Ticagrelor was purchased from Oxchem Corporation (Wood Dale, IL,
USA). AYPGKF-NH2 was obtained from Abgent (San Diego, CA, USA). Oregon Green® 488 BAPTA-1 AM (OG488 BAPTA-1 AM)
was from Thermo Fisher Scientific (Carlsbad, CA, USA). PAC-1-FITC and anti-CD62P-APC were obtained from BD Bioscience (San Jose,
CA, USA). Anti-AKT, phospho (p)AKT-Ser473, pGSK3β-Ser9 and pPKC-Ser substrates were from Cell Signaling Technology
(Danvers, MA, USA).
Mitrugno A., Rigg R.A., Laschober N.B., Ngo A.T., Pang J., Williams C.D., Aslan J.E, & McCarty O.J. (2017). Potentiation of TRAP-6-induced platelet dense granule release by blockade of P2Y12 signaling with MRS2395. Platelets, 29(4), 383-394.
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2

Platelets Adhesion and Activation Assay

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Fibrillar collagen (Horm suspension) was from Nycomed (Munich, Germany). Alexa Fluor® 568 conjugated annexin V (annexin-V; anxV) and Fluo-4 AM, were purchased from Life Technologies. Alexa Fluor® 568-Labelled Lactadherin was purchased from Cambridge Bioscience Ltd. Bovine serum albumin (BSA), ADP, Aspirin and Apyrase were from Sigma Aldrich. RWJ-56110, MRS 2279 and AR-C66096 were from Tocris (Bristol, UK). PPACK was from Enzo life science. Connexin inhibitors, Cx37Gap27 (SRPTEKTIFII) was from Sigma Aldrich and Cx40Gap27 (SRPTEKNVFIV) was from Anaspec, USA. Glass bottom 35 mm dishes (P35G-1.5-20-C) were obtained from MatTek Corporation (USA). Flow chambers (Vena8TM biochip) were from Cellix Ltd. The fluorogenic thrombin substrate Z-Gly-Gly-Arg aminomethyl coumarin (Z-GGR-AMC) was from Bachem AG. Dylight 488–conjugated anti-GPIbβ antibody was from Emfret Analytics, Eibelstadt, Germany.
Agbani E.O., Williams C.M., Hers I, & Poole A.W. (2017). Membrane Ballooning in Aggregated Platelets is Synchronised and Mediates a Surge in Microvesiculation. Scientific Reports, 7, 2770.
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3

Pharmacological Modulation of P2Y Receptors

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All chemicals were purchased from Sigma-Aldrich (UK) with the exception of BMSCCR222, BAPTA AM, MRS2578, ARL67156, AR-C66096 and Bordetella pertussis toxin (PTx; Tocris Bioscience, UK), MRS2179, MRS2211 (Abcam Biochemicals, UK), CCL2, TNFα and Fluo-4 AM (Life Technologies, UK), and Calcein AM (Santa Cruz Biotechnology, Germany). THP-1 and HEK 293T cells were procured from the European Collection of Cell Cultures (ECACC), and HUVECs from Caltag Medsystems (UK). Human 1321N1 P2Y6 stable cells were a kind gift from Jens Leipziger (Aarhus University, Denmark). Compounds used did not induce toxicity under assay conditions as determined by a Trypan Blue exclusion assay or lactate dehydrogenase (LDH) release assay.
Campwala H., Sexton D.W., Crossman D.C, & Fountain S.J. (2014). P2Y6 receptor inhibition perturbs CCL2-evoked signalling in human monocytic and peripheral blood mononuclear cells. Journal of Cell Science, 127(22), 4964-4973.
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4

Pretreatment and LPS Stimulation Assay

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Cells were pretreated for 1 h with drug treatment. ITI-214 was synthesized at Intra-Cellular Therapies, Inc. (Li et al., 2016 (link)). Other drugs were purchased from Sigma-Aldrich (ADP, IBMX, rolipram) or Tocris (AR-C 66096, and AR-C 69931). Where indicated, following pretreatment, LPS (from E. coli 0111:B4, Millipore-Sigma) was added to the media at 50 ng/mL for 4 h. At 5 h, the cells or media were harvested and processed as indicated below.
O'Brien J.J., O'Callaghan J.P., Miller D.B., Chalgeri S., Wennogle L.P., Davis R.E., Snyder G.L, & Hendrick J.P. (2019). Inhibition of calcium-calmodulin-dependent phosphodiesterase (PDE1) suppresses inflammatory responses. Molecular and cellular neurosciences, 102, 103449.
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5

Flow Chamber Assay Materials and Reagents

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The cover and capillary chips used in the flow chamber system (Fig. S1A) were manufactured by Richell Corp. (Toyama, Japan). The following materials were obtained from commercial sources: porcine type I collagen (Nitta Gelatin, Inc., Osaka, Japan), tissue thromboplastin (Sysmex, Hyogo, Japan), fluorescein isothiocyanate (FITC)-conjugated mouse anti-human CD41 immunoglobulin G (IgG), and FITC-conjugated mouse IgG (Beckman Coulter, Miami, FL, USA), rabbit anti-human fibrinogen IgG (Dako, Tokyo, Japan), normal rabbit IgG (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and Alexa594 (Invitrogen, Carlsbad, CA, USA).
Dabigatran and rivaroxaban were obtained from Toronto Research Chemicals, Inc. (Toronto, Canada). AR-C66096, a specific P2Y12-receptor antagonist, was obtained from Tocris Bioscience (Bristol, UK). For the TG assay, PPP-Reagent (with phospholipids), PRP-Reagent (without phospholipids), and FluCa-reagent, a fluorogenic substrate (Z-Gly-Gly-Arg-AMC) dissolved in HEPES buffer and calcium chloride, were purchased from Diagnostica Stago (Parsippany, NJ). Recombinant TF (r-TF) was purchased from Mitsubishi Chemical Medience (Tokyo, Japan). All other reagents were obtained from Wako Pure Chemicals (Osaka, Japan). Corn trypsin inhibitor (CTI) was prepared as reported previously [13] (link).
Hosokawa K., Ohnishi T., Sameshima H., Miura N., Koide T., Maruyama I, & Tanaka K.A. (2014). Comparative Evaluation of Direct Thrombin and Factor Xa Inhibitors with Antiplatelet Agents under Flow and Static Conditions: An In Vitro Flow Chamber Model. PLoS ONE, 9(1), e86491.
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6

Pretreatment and LPS Stimulation Assay

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Cells were pretreated for 1 h with drug treatment. ITI-214 was synthesized at Intra-Cellular Therapies, Inc. (Li et al., 2016 (link)). Other drugs were purchased from Sigma-Aldrich (ADP, IBMX, rolipram) or Tocris (AR-C 66096, and AR-C 69931). Where indicated, following pretreatment, LPS (from E. coli 0111:B4, Millipore-Sigma) was added to the media at 50 ng/mL for 4 h. At 5 h, the cells or media were harvested and processed as indicated below.
O'Brien J.J., O'Callaghan J.P., Miller D.B., Chalgeri S., Wennogle L.P., Davis R.E., Snyder G.L, & Hendrick J.P. (2019). Inhibition of calcium-calmodulin-dependent phosphodiesterase (PDE1) suppresses inflammatory responses. Molecular and cellular neurosciences, 102, 103449.
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7

Platelet Function Assay Reagents

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All reagents were from Sigma unless otherwise stated. AR-C66096 and eptifibatide were from Tocris (Bristol, U.K.). PAR1-AP (SFLLRN-amide) and PAR4-AP (AYPGKF-amide) were from Bachem (Weil-am-Rhein, Germany). Cross-linked collegen-related peptide (CRP-XL) was synthesised by Arkadiusz Bonna, Department of Biochemistry. Function blocking antibodies were preservative-free. The antibody clones are listed in Supplementary Table 1.
Ivanov I.I., Apta B.H., Bonna A.M, & Harper M.T. (2019). Platelet P-selectin triggers rapid surface exposure of tissue factor in monocytes. Scientific Reports, 9, 13397.
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