P2Y12 receptor

Inhibition of P2Y12 by MRS2395 enhances TRAP-6-induced platelet dense granule release. Inhibition of P2Y12 with MRS2395 enhances the localization of dense granules at the plasma membrane after platelet activation with TRAP-6. MRS2395-dependent potentiation of TRAP-6-induced dense granule secretion is PI3K-independent and PLC-dependent. MRS2395-dependent potentiation of TRAP-6-induced dense granule secretion is PKC and calcium-dependent. Glycogen Synthase Kinase 3β (GSK3β) plays a role in the ability of MRS2395 to potentiate platelet activation downstream of the PAR-1 receptor [2].

Imaging of dense granule intracellular localization and reorganization by Superresolution Structured Illumination Microscopy (SR-SIM). [2]
For imaging experiments, 12mm #1.5 glass coverslips were coated with poly-L-lysine. Inhibitors (MRS2395, 10 μM; ticagrelor, 20 ng/mL; PSB 0739, 10 μM; AR-C 66096, 10 μM) were added to platelets in solution (4×107/mL) for 15 min prior to activation with TRAP-6 (10 μM) for 30 sec at 37ºC, followed by fixation with 4% paraformaldehyde and seeding onto proteins at room temperature for 1 hr. Adherent platelets were permeabilized with a blocking solution (1% BSA + 0.01% SDS in PBS). Platelets were then stained with CD63 (MX49.129.5) and MRP4/ABCC4 (D1Z3W) overnight at 4ºC at a 1:100 dilution in the blocking solution. Alexa Fluor secondary antibodies (1:500) were added in the blocking solution for 2 hrs. Coverslips were mounted with Fluoromount G onto glass slides. Platelets were imaged using SR-SIM with a Zeiss 100× oil immersion 1.46 NA alpha plan-apochromat lens on a Zeiss Elyra PS.1 microscope.

Flow cytometry analysis. [2]
Flow cytometry experiments were carried out as previously described. Briefly, washed human platelets (2×108 /mL) were preincubated with vehicle (0.01% DMSO) or MRS2395 (10 μM) for 15 min at 37°C and then stimulated with ADP (10 μM final concentration) for 15 min in the presence of FITC-conjugated anti-human PAC-1 or APC-conjugated anti-human CD62P antibodies. Samples were then fixed by 1% paraformaldehyde for 10 min and diluted in HEPES/Tyrode buffer, followed by flow cytometric analysis on a FACSCantoII system. Data are expressed as percentage of the maximal response obtained in the response to ADP.

[1]. Acyclic analogues of adenosine bisphosphates as P2Y receptor antagonists: phosphate substitution leads to multiple pathways of inhibition of platelet aggregation. J Med Chem. 2002 Dec 19;45(26):5694-709.

[2]. Potentiation of TRAP-6-induced platelet dense granule release by blockade of P2Y 12 signaling with MRS2395. Platelets. 2018 Jun;29(4):383-394.

ADP activation of P2Y(1) and P2Y(12) receptors in platelets promotes platelet aggregation, and antagonists of these receptor subtypes have antithrombotic effects. In a previous study, we characterized the structure-activity relationship (SAR) of P2Y(1) receptor antagonists as adenosine nucleotide acyclic analogues containing two phosphate groups attached to the 9-position of adenine on a symmetrically branched fatty chain. In this study, we focus on the antiaggregation effects of P2Y antagonists associated with the 2-chloro-N(6)-methyladenine-9-(2-methylpropyl) backbone, in which the phosphate groups are uncharged. For known P2Y(1) receptor nucleotide (cyclic and acyclic) diphosphate antagonists, a significant correlation was found between 3.3 μM ADP-induced aggregation inhibition in rat platelets and previously measured P2Y(1) receptor-induced inhibition of phospholipase C (PLC) activity in turkey erythrocytes. Replacing phosphate groups with non-hydrolyzable phosphonate groups preserves platelet anti-aggregation activity. Replacing one phosphate group with an O-acyl group significantly reduces inhibitory efficacy, while replacing both phosphate groups in acyclic derivatives with uncharged groups (e.g., ester groups) tends to enhance inhibitory efficacy. Among a range of asymmetrically substituted monophosphates, the phenylcarbamate group exhibits the best antagonistic efficacy. Among symmetric diester derivatives, the di(phenylacetyl) group shows the best antagonistic efficacy. A divalveyl derivative (a representative uncharged diester) inhibits ADP-induced aggregation in rat (K_I 3.6 μM) and human platelets. It antagonizes ADP-induced inhibition of the rat platelet cyclic adenosine monophosphate (cAMP) pathway (IC₅₀ 7 μM) but does not affect hP2Y₁ receptor-induced phospholipase C (PLC) activity in transfected astrocytoma cells. We hypothesize that these uncharged derivatives are antagonists of another platelet aggregation receptor, namely the P2Y₁₂ receptor. Thus, different substituents of the same nucleoside backbone can target one of the two P2Y receptors in platelets. [1]

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The release of ADP from platelet dense granules and its binding to platelet P2Y₁₂ receptors is key to enhancing the initial hemostatic response and promoting thrombosis. Therefore, P2Y12 has become a safe and effective therapeutic target for the prevention of secondary thrombotic events in patients with acute coronary syndrome or previous myocardial infarction. Pharmacological inhibition of the P2Y12 receptor is an effective way to gain a deeper understanding of the signaling pathways mediated by these receptors and to elucidate the role of these receptors in various platelet hemostatic and thrombotic responses. This study investigated and compared the effects of four different P2Y12 inhibitors (MRS2395, ticagrelor, PSB 0739, and AR-C 66096) on platelet function, including the secretion and transport of platelet dense granules, calcium ion production, and protein phosphorylation, through a series of in vitro experiments. Our results indicate that in platelets activated by the PAR-1 agonist TRAP-6 (thrombin receptor activating peptide), inhibition of P2Y12 using the P2Y12 antagonist MRS2395 (rather than ticagrelor, PSB 0739, or AR-C 66096) enhances the transport of human platelet dense granules to the plasma membrane and their release into the extracellular space, promotes cytoplasmic Ca2+ influx, and enhances GSK3β-Ser9 phosphorylation via a PKC-dependent pathway. These results suggest that the inhibition of P2Y12 by MRS2395 may synergize with the PAR-1 signaling pathway, leading to abnormal release of ADP from platelet dense granules, thereby reducing or counteracting the expected antiplatelet activity of the inhibitor. [2]

C20H30CLN5O4

439.93630361557

439.199

491611-55-3

10071919

Typically exists as solid at room temperature

3.389

1

8

11

30

577

0

CC(C)(C)C(=O)OCC(CN1C=NC2=C(NC)N=C(Cl)N=C21)COC(=O)C(C)(C)C

NASABYJQIYJDID-UHFFFAOYSA-N

InChI=1S/C20H30ClN5O4/c1-19(2,3)16(27)29-9-12(10-30-17(28)20(4,5)6)8-26-11-23-13-14(22-7)24-18(21)25-15(13)26/h11-12H,8-10H2,1-7H3,(H,22,24,25)

[2-[[2-chloro-6-(methylamino)purin-9-yl]methyl]-3-(2,2-dimethylpropanoyloxy)propyl] 2,2-dimethylpropanoate

491611-55-3; MRS 2395; mrs2395; 2,2-DIMETHYL-PROPIONIC ACID 3-(2-CHLORO-6-METHYLAMINOPURIN-9-YL)-2-(2,2-DIMETHYL-PROPIONYLOXYMETHYL)-PROPYL ESTER; CHEMBL347921; [2-[[2-chloro-6-(methylamino)purin-9-yl]methyl]-3-(2,2-dimethylpropanoyloxy)propyl] 2,2-dimethylpropanoate; SCHEMBL690985; DTXSID40435170;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)