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| Targets |
PAR1
Parstatin targets the tethered ligand domain of the PAR-1 thrombin receptor, mimicking the activated receptor's internally bound ligand after proteolytic cleavage by thrombin. While classical PAR-1 activation by thrombin leads to G-protein signaling (Galphaq/11, Galpha12/13), Parstatin acts as a biased agonist, triggering cytoprotective and anti-angiogenic pathways (likely Galphai-mediated) without inducing the full pro-inflammatory and pro-thrombotic effects of thrombin. |
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| ln Vitro |
Parstatin (zero to ten micrograms) enhances LVDP recovery in a concentration-dependent way. The best concentration was 1 µM, which resulted in a 23% LVDP recovery[2].
In assays of angiogenesis, Parstatin (1-10 uM) potently inhibits human umbilical vein endothelial cell (HUVEC) proliferation, migration, and capillary-like tube formation on Matrigel in a concentration-dependent manner. It blocks vascular endothelial growth factor (VEGF)-induced endothelial cell sprouting and reduces the secretion of matrix metalloproteinases (MMP-2 and MMP-9). In primary mouse endothelial cells, Parstatin enhances LVDP recovery. |
| ln Vivo |
Administering parstatin (single dosage, 1-25 µg/kg, IV) prior to ischaemia results in rapid cardioprotection through the activation of the Gi-protein activation pathway, which includes p38 MAPK, ERK1/2, NOS, and KATP channels. To produce cardioprotection, parstatin acts on the coronary circulation as well as the cardiomyocytes. This implies that parstatin may have a therapeutic use in the management of heart damage brought on by reperfusion and ischaemia[1].
In vivo, Parstatin (1-10 nmol, injected locally or systemically) reduces angiogenesis in the mouse Matrigel plug assay and in the chick chorioallantoic membrane (CAM) assay. In a mouse model of hindlimb ischemia, Parstatin administration limits pathological neovascularization. In models of myocardial infarction, it improves left ventricular developed pressure (LVDP) recovery. It significantly inhibits tumor growth in syngeneic mouse models by reducing tumor vascularization. |
| Enzyme Assay |
In vitro enzyme/receptor binding studies utilize a competition binding assay. Membranes from CHO or HEK293 cells overexpressing mouse PAR-1 are prepared by homogenization and differential centrifugation. Membranes (50 ug protein) are incubated with 5 nM [3H]haTRAP (a radioactive PAR-1 activating peptide) or ¹2⁵I-labeled thrombin as a radioligand, and increasing concentrations (0.1 nM - 10 uM) of unlabeled Parstatin(mouse) TFA or mouse Parstatin peptide in binding buffer (25 mM HEPES, pH 7.4, 10 mM MgCl2, 0.1% BSA) for 60-120 min at 25degC. Bound radioligand is separated by rapid vacuum filtration through GF/C filters pre-soaked in 0.5% BSA. Filters are washed 3 times with ice-cold binding buffer and counted by liquid scintillation. Nonspecific binding is defined using 10 uM unlabeled Parstatin. Data are analyzed by nonlinear regression to calculate IC50 and Ki.
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| Cell Assay |
GTPgammaS binding assays measure Parstatin-induced G protein activation. Membranes from mouse PAR-1-expressing cells (50 ug protein) are preincubated with varying concentrations (1 nM - 10 uM) of Parstatin in assay buffer (20 mM HEPES, pH 7.4, 100 mM NaCl, 10 mM MgCl2, 1 mM EDTA, 1 mM DTT, 1 uM GDP) for 15 min at 30degC. Then 0.1 nM [3⁵S]GTPgammaS is added, and the reaction is incubated for an additional 30 min. Bound radioactivity is separated by filtration through GF/B filters. Counts are measured by scintillation counting. The EC50 is calculated. For anti-angiogenesis studies, HUVECs (passages 3-6) are cultured in EGM-2 media. For proliferation, cells (2×103 cells/well) are seeded in 96-well plates in EGM-2 with 0.5% FBS. After 24 h, they are treated with Parstatin (0.1-10 uM) or control peptide in EGM-2 plus 10 ng/mL VEGF for 48-72 h. Cell viability is measured by MTT (0.5 mg/mL, 4 h). For migration, a scratch assay is performed: confluent HUVEC monolayers (in 6-well plates) are scratched with a 200 microL pipette tip, washed, and treated with Parstatin (1-10 uM) with 10 ng/mL VEGF for 24 h. Wound closure is quantified by microscopy (ImageJ). For tube formation, HUVECs (2×10⁴ cells/well) are seeded on 50 uL/well growth factor-reduced Matrigel (Corning) in 96-well plates. Cells are treated with Parstatin (1-10 uM) and VEGF (10 ng/mL) for 6-18 h. Tube length and number of nodes are quantified. Apoptosis is assessed by Hoechst 33342 staining and cleaved caspase-3 Western blot.
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| Animal Protocol |
Animal/Disease Models: Male Sprague–Dawley rats at 8 weeks of age (250-300 g) [1].
Doses: 1-25 µg/kg. Route of Administration: IV injected 15 min prior to ischaemia. Experimental Results: A significant decrease in infarct size was detected with the 5-15 µg/kg doses with 10 µg/kg as the optimal dose. These hearts had an infarct size of 46 ± 3% of the area at risk, which is a 26% reduction in infarct size compared with the control. In a mouse hindlimb ischemia model, 8-10 week old C57BL/6J mice (n=8-10 per group) undergo unilateral femoral artery ligation and excision. Immediately after surgery, Parstatin(mouse) TFA (dissolved in 0.1% BSA in saline) is injected intramuscularly at the ischemic site at doses of 0.1, 0.5, or 1 ug/kg (in 100 uL) and repeated every 48 hours for 14 days. Blood flow recovery is assessed by laser Doppler perfusion imaging (LDPI) at days 0, 3, 7, and 14, with the ratio of ischemic to non-ischemic limb blood flow calculated. At day 14, gastrocnemius muscles are collected for capillary density assessment by CD31 immunofluorescence staining. In a myocardial I/R model (mouse: 30 min LAD occlusion, 24 h reperfusion), Parstatin (1-50 ug/kg, IV bolus) is administered 5 min before reperfusion. Infarct size is measured by TTC/Evans blue staining. Cardiac function is assessed by echocardiography. |
| ADME/Pharmacokinetics |
Pharmacokinetic data for Parstatin are not published. As a 41-amino acid peptide, systemic half-life is expected to be very short (<15 minutes) due to rapid proteolytic degradation. For in vivo studies, continuous infusion or frequent daily dosing may be required to maintain therapeutic levels. Local administration (intramuscular for hindlimb ischemia) bypasses rapid systemic clearance. The TFA salt provides water solubility.
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| Toxicity/Toxicokinetics |
A comprehensive toxicity profile is lacking. Parstatin is derived from the natural PAR-1 sequence and, as a cell-penetrating peptide, is generally well-tolerated at the doses used in animal studies (<1 mg/kg). No overt signs of systemic toxicity, behavioral changes, or organ damage are observed at doses up to 5 mg/kg in mice. It does not induce excessive bleeding or thrombosis, as it does not activate PAR-1's pro-coagulant pathways. High-dose toxicity has not been systematically evaluated.
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| References |
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| Additional Infomation |
Parstatin is a research-grade peptide and has not been approved for clinical use. It belongs to a family of "tethered ligand" peptides derived from PARs (including PAR-1, PAR-2, PAR-3, and PAR-4). This mouse-specific sequence shares ~71% identity with human Parstatin. Parstatin represents a novel class of angiogenesis inhibitors that act via GPCRs. The TFA salt form is standard for peptide research.
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| Molecular Formula |
C191H327F3N58O59S3
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| Molecular Weight |
4533.18
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| Related CAS # |
Parstatin(mouse);1065756-01-5
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| Appearance |
White to off-white solid powder
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| HS Tariff Code |
2934.99.9001
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| Storage |
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. |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
H2O :~100 mg/mL (~22.06 mM)
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| Solubility (In Vivo) |
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.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *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). View More
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)] Oral Formulations
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). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 0.2206 mL | 1.1030 mL | 2.2060 mL | |
| 5 mM | 0.0441 mL | 0.2206 mL | 0.4412 mL | |
| 10 mM | 0.0221 mL | 0.1103 mL | 0.2206 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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