The primary target of PAR-4 (1-6) amide (human) is the protease-activated receptor 4 (PAR4), a G protein-coupled receptor (GPCR) expressed on platelets, endothelial cells, and other cell types. PAR4 is activated by proteolytic cleavage of its extracellular N-terminal domain by serine proteases such as thrombin, revealing a tethered ligand that binds to the receptor's second extracellular loop. The synthetic peptide PAR-4 (1-6) amide mimics this tethered ligand and directly activates the receptor without the need for proteolytic cleavage. Activation of PAR4 primarily triggers Galpha12/13 and Galphaq signaling pathways, leading to Rho activation, cytoskeletal rearrangement, and platelet shape change, as well as activation of phospholipase C (PLC) and subsequent calcium mobilization. In human platelets, PAR4 activation results in robust aggregation and secretion, particularly at high thrombin concentrations, and plays an important role in the amplification of the thrombin response. The amidation at the C-terminus increases the peptide's stability and resistance to carboxypeptidases.

Platelet aggregation is induced by human PAR-4 (1-6) amide at 50 and 100 µM [1].

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In vitro studies demonstrate that PAR-4 (1-6) amide (human) at concentrations of 50 and 100 uM induces platelet aggregation. The peptide activates PAR4 in a thrombin-independent manner, making it a valuable tool for studying the specific role of PAR4 in platelet function. In platelet-rich plasma (PRP) or washed human platelet suspensions, the addition of PAR-4 (1-6) amide results in a concentration-dependent increase in platelet aggregation, as measured by light transmission aggregometry (LTA). The aggregation is accompanied by activation of the fibrinogen receptor alphaIIbbeta3 (GPIIb/IIIa), as measured by flow cytometry using PAC-1 antibody, and release of granular contents (e.g., ATP, serotonin). The peptide also induces an increase in intracellular calcium levels ([Ca2+]i) in platelets, as measured by Fluo-3 or Fura-2 fluorescence, and activates downstream signaling pathways, including phosphorylation of Akt (Ser473) and ERK1/2 (Thr202/Tyr204). Unlike PAR1-activating peptides (e.g., SFLLRN), PAR-4 (1-6) amide is a weaker agonist and requires higher concentrations (50-100 uM) to achieve maximal aggregation, reflecting the lower sensitivity of PAR4 compared to PAR1 in human platelets. The peptide can also desensitize PAR4, preventing subsequent activation by thrombin or other PAR4 agonists. In other cell types expressing PAR4, such as endothelial cells, the peptide may induce calcium signaling and barrier dysfunction.

In vivo studies for PAR-4 (1-6) amide are not typically performed, as the peptide is an agonist and would likely induce systemic platelet activation and thrombosis if administered intravenously. It is primarily an in vitro tool for studying platelet and PAR4 biology. However, for research purposes, the peptide could be injected intravenously into animals (e.g., mice) at low doses to study its effects on platelet activation and thrombus formation in vivo. For example, in a mouse model of thrombosis (e.g., FeCl3-induced carotid artery injury), intravenous administration of PAR-4 (1-6) amide (e.g., 1-10 mg/kg) prior to injury could potentially accelerate thrombus formation or reduce the time to occlusion. Alternatively, it could be used in a tail-bleeding model to assess the effect on hemostasis. These studies would help elucidate the role of PAR4 in in vivo thrombosis and hemostasis. However, no specific in vivo data is available in the search results. Given the species differences in PAR4 sequence (mouse PAR4 is not activated by human PAR-4-activating peptides), such studies would require humanized PAR4 mouse models or the use of species-specific PAR4 agonists.

Non-cell-based assays for PAR-4 (1-6) amide are not typical, as it is a peptide agonist that activates a GPCR and its activity is measured in cell-based functional assays. However, one could use radioligand binding assays using membranes from cells expressing human PAR4 (e.g., HEK293T cells transiently transfected with PAR4). Membranes (20-50 ug protein) are incubated with a radiolabeled PAR4 antagonist (e.g., 3H-YD-3, if available) and increasing concentrations of unlabeled PAR-4 (1-6) amide (0.1 nM to 100 uM) in binding buffer (50 mM Tris-HCl pH 7.4, 10 mM MgCl2, 1 mM EDTA, 0.1% BSA) for 60 minutes at room temperature. Non-specific binding is determined in the presence of 10 uM unlabeled agonist. Bound ligand is separated by rapid filtration through GF/C filters (presoaked in 0.3% polyethylenimine) and counted. The IC50 for displacement of the radioligand is determined, and the Ki is calculated. Alternatively, the direct binding of a fluorescently labeled peptide (e.g., GYPGQV-FAM) could be measured by fluorescence polarization (FP) or TR-FRET. However, these assays are not routine. For a functional cell-free assay, a membrane-based GTPgammaS binding assay can be performed (see below for cell-based version). Membranes (20-30 ug) from PAR4-expressing cells are incubated with 0.1 nM 35S-GTPgammaS, 100 uM GDP, and varying concentrations of PAR-4 (1-6) amide (0.1-100 uM) in assay buffer (20 mM HEPES pH 7.4, 100 mM NaCl, 5 mM MgCl2) for 60 minutes at 25degC. Bound radioactivity is separated by filtration and counted. The EC50 for stimulation of GTPgammaS binding is calculated. This assay directly measures G protein activation by the agonist-bound receptor.

For cell-based studies, human platelets are isolated from fresh whole blood. Blood is collected into 3.2% sodium citrate (9:1) and centrifuged at 200g for 10 minutes at room temperature to obtain platelet-rich plasma (PRP). The PRP is diluted with autologous platelet-poor plasma (PPP, obtained by further centrifugation at 2000g for 10 minutes) to achieve a standard platelet count (e.g., 2.5×10^8 platelets/mL). For washed platelets, PRP is acidified with ACD (85 mM trisodium citrate, 70 mM citric acid, 110 mM dextrose, pH 6.5) and centrifuged at 1000g for 10 min. The platelet pellet is resuspended in Tyrode's buffer (pH 7.4) with 1 mM CaCl2 and 1 mM MgCl2. Platelets are then activated by adding PAR-4 (1-6) amide (e.g., 0, 10, 50, 100, 200, 500 uM) and stirred at 37degC for 5-10 minutes in a lumi-aggregometer. Aggregation is measured as the increase in light transmission relative to PPP (100% transmission). ATP release is measured by the luciferin/luciferase method (Chrono-Lume reagent). For calcium mobilization assays, washed platelets are loaded with Fluo-3 AM (5 uM) or Fura-2 AM (2 uM) for 30 minutes at 37degC in the dark, diluted to 2×10^8/mL, and then stimulated with the peptide. Fluorescence changes (λex 485 nm, λem 525 nm for Fluo-3) are measured in a fluorescence plate reader or spectrofluorometer. For flow cytometry analysis of platelet activation, PRP or washed platelets are stimulated with the peptide, then fixed with 0.5% paraformaldehyde, and stained with fluorescently labeled antibodies against CD62P (P-selectin; for alpha-granule release), CD63 (lysosomal membrane protein), or PAC-1 (activated alphaIIbbeta3). Samples are analyzed on a flow cytometer. For signaling studies (e.g., Western blotting), platelets are activated with PAR-4 (1-6) amide (e.g., 100 uM) for 0, 1, 2, 5, 10 minutes, then lysed in RIPA buffer with phosphatase inhibitors, and immunoblotted with antibodies against phospho-Akt (Ser473), phospho-ERK1/2 (Thr202/Tyr204), or phospho-PLCgamma2 (Tyr1217). For studies in other cell types (e.g., HEK293T cells transiently expressing human PAR4), cells are seeded in 96-well plates (1×10^4 cells/well) and loaded with the calcium indicator Fluo-4 AM (2 uM) for 30 minutes. Increasing concentrations of the peptide are added, and the fluorescence intensity is measured (λex 485 nm, λem 525 nm) using a plate reader. The EC50 for calcium mobilization is calculated. PAR4-mediated calcium signaling can be blocked by specific PAR4 antagonists (e.g., YD-3, ML354) or by PAR4-targeting siRNAs.

In vivo protocols for PAR-4 (1-6) amide are not standard, but for research purposes, it could be administered to animal models of thrombosis. For example, in a mouse model of FeCl3-induced carotid artery injury, male C57BL/6 mice (8-10 weeks old, 20-25 g) are anesthetized, and the carotid artery is exposed. A small piece of filter paper soaked in 10% FeCl3 is applied to the artery for 2-3 minutes to induce vessel injury and thrombus formation. PAR-4 (1-6) amide (human) is not expected to activate mouse PAR4 due to species differences (mouse PAR4 has a different tethered ligand sequence). Therefore, a genetically modified mouse expressing human PAR4 (humanized PAR4 mouse) would be required. In such a model, the peptide (e.g., 0.1-10 mg/kg) could be administered via intravenous (i.v.) injection 5-10 minutes before FeCl3 injury. Blood flow is monitored using a Doppler flow probe, and the time to occlusion (complete cessation of blood flow) is measured. The peptide might shorten the time to occlusion, indicating a pro-thrombotic effect. Alternatively, in a tail bleeding model, mice are anesthetized, and a 2 mm segment of the tail tip is amputated. The tail is immersed in saline at 37degC, and the bleeding time (the time to cessation of bleeding) is measured. Administration of PAR-4 (1-6) amide might shorten bleeding time, indicating a pro-hemostatic effect. Dosing would be based on pilot studies, typically 0.1-5 mg/kg i.v. These studies are not intended for drug development but rather to study the role of PAR4 in thrombus formation in a system that mimics human PAR4 biology.

Pharmacokinetic data for PAR-4 (1-6) amide is not available, as it is a research peptide used for in vitro studies. As a hexapeptide (MW 618.68 Da), it is susceptible to rapid proteolytic degradation by proteases in plasma and tissues. The half-life in circulation, if administered intravenously, would be on the order of minutes (e.g., 2-10 minutes). The compound would likely be rapidly cleaved by exopeptidases and endopeptidases into smaller peptides and free amino acids. The amide group at the C-terminus provides some protection against carboxypeptidases, but it is still vulnerable to other proteases. The peptide is relatively polar and would have low oral bioavailability (<1-5%). For in vitro assays, stock solutions are prepared in water or DMSO (e.g., 100 mM). The lyophilized peptide should be stored at -20degC, protected from light and moisture, and is stable for at least 2 years. In solution, it should be stored at -80degC in aliquots and used within 6 months to avoid degradation.

Preclinical toxicity data for PAR-4 (1-6) amide is not available, as it is not a drug candidate. In cell-based assays (platelets, HEK293 cells), the peptide does not cause direct cytotoxicity at concentrations up to 500 uM. It is a specific agonist of PAR4 and does not activate other PAR family members (PAR1, PAR2, PAR3) at the concentrations used. Systemic administration in vivo, as described in hypothetical studies, would likely lead to acute platelet activation, thrombocytopenia (low platelet count), and potentially thrombotic events (e.g., pulmonary embolism, stroke). Therefore, it is not safe for human use. Standard safety precautions for handling peptides (gloves, lab coat, goggles) should be followed. The compound is for research use only and is not for human or therapeutic use. No genotoxicity, carcinogenicity, or reproductive toxicity studies have been conducted.

[1]. Shimizu M, et al. Thrombin-induced platelet aggregation -effect of dabigatran using automated platelet aggregometry. Platelets. 2020;31(3):360-364.

PAR-4 (1-6) amide (human) is a synthetic peptide agonist of the human protease-activated receptor 4 (PAR4). It is commonly used as a research tool to activate PAR4 in vitro and to study the role of PAR4 in platelet aggregation, thrombosis, and other cellular processes. The peptide is also known as GYPGQV-NH2, and its sequence is derived from the N-terminal region of the human PAR4 receptor that is exposed after thrombin cleavage. It is not a drug and has no FDA-approved indications. It is soluble in water (e.g., 90 mg/mL) and DMSO. The product should be stored as a lyophilized powder at -20degC, protected from light and moisture, and sealed in a desiccated container. Under these conditions, it is stable for at least 2 years. In solution, it should be stored in aliquots at -80degC and used within 6 months. This peptide is an essential tool for hemostasis and thrombosis research.

C28H42N8O8

618.68

618.312

245443-51-0

131851703

White to off-white solid powder

1.5±0.1 g/cm3

913.8±75.0 °C at 760 mmHg

506.5±37.1 °C

0.0±0.3 mmHg at 25°C

1.656

0.98

8

9

16

44

1060

4

C(N)(=O)[C@H](C(C)C)NC(=O)[C@H](CCC(N)=O)NC(=O)CNC(=O)[C@@H]1CCCN1C(=O)[C@H](CC1=CC=C(O)C=C1)NC(=O)CN

NIUWXNGGQOLCDN-XHOYROJHSA-N

InChI=1S/C28H42N8O8/c1-15(2)24(25(31)41)35-26(42)18(9-10-21(30)38)33-23(40)14-32-27(43)20-4-3-11-36(20)28(44)19(34-22(39)13-29)12-16-5-7-17(37)8-6-16/h5-8,15,18-20,24,37H,3-4,9-14,29H2,1-2H3,(H2,30,38)(H2,31,41)(H,32,43)(H,33,40)(H,34,39)(H,35,42)/t18-,19-,20-,24-/m0/s1

(2S)-2-[[2-[[(2S)-1-[(2S)-2-[(2-aminoacetyl)amino]-3-(4-hydroxyphenyl)propanoyl]pyrrolidine-2-carbonyl]amino]acetyl]amino]-N-[(2S)-1-amino-3-methyl-1-oxobutan-2-yl]pentanediamide

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light.

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

H2O: 100 mg/mL (161.63 mM)

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.)