PAR1/rotease activating receptor 1 (IC50 = 96 nM)

Calcium mobilization in Xenopus laevis oocytes heterologously expressing PAR1 is triggered by TRAP-6 (0.01-10 μM) [1]. Human platelets are activated for 30 minutes by TRAP-6 (0.01-10 μM) [1]. In rats or rabbits, TRAP-6 (100 μM) did not cause platelets to aggregate, discharge granule contents, change shape, or create thromboxane [2].

In actin-anesthetized rats, TRAP (1 mg/kg; i.v.) causes a biphasic blood pressure response [3].

In vitro platelet aggregation in rat PRP [3]
Male rats (250–300 g) were anesthetized with inactin (100 mg/kg, i.p.). After an abdominal incision, the aorta was exposed and entered just anterior to the bifurcation with a 21G Vacutainer multiple-sample needle. Donor blood (9 ml) was collected in two citrate Vacutainer tubes (containing 0.5 ml of 3.2% buffered sodium citrate solution). After centrifugation (130×g for 15 min), platelet rich plasma was removed and used for the aggregation assay as described in Section 2.1.1. In those studies where amastatin was used the platelets were incubated with amastatin for 2 min before challenge with the agonist. To determine if pretreatment with TRAP causes desensitization of thrombin induced aggregation, rat platelet rich plasma was incubated with 100 μM TRAP for 5 min at 37°C before challenge with 0.1 U/ml of thrombin.

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Aggregation assay [3]
Platelet aggregation was performed in a dual channel Chronolog aggregometer. Briefly, 0.48 ml of platelet rich plasma was added to the cuvettes and incubated at 37°C for 5 min. Aggregation was initiated by addition of human TRAP or rat peptide (SFFLRN) or thrombin to the platelets and the aggregation response was then monitored for 5 min on an IBM computer and the peak aggregation response was determined turbidimetrically with the help of the Aggro/LINK software.

In vivo blood pressure responses in rats [3]
Rats were anesthetized and prepared for intravenous injections as described above. In addition the left carotid artery was cannulated (PE-50) and blood pressure was recorded with a Statham pressure transducer connected to a Grass polygraph. In the nephrectomized rats a lateral midline incision was made, the renal arteries isolated and a silk suture was passed around the vessel to facilitate ligature. After a 30 min equilibration period one of the following experiments were carried out.

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Effects of TRAP on blood pressure [3]
Vehicle (0.1 ml saline) or TRAP (1 mg/kg, i.v. bolus) were administered i.v. bolus and the changes in blood pressure were monitored for 30 min.

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Effect of NG-nitro l-arginine methyl ester (l-NAME) on the blood pressure response to TRAP [3]
A control blood pressure response to TRAP (1 mg/kg, i.v.) was obtained. When the blood pressure returned to baseline intravenous infusion of l-NAME (0.3 mg/kg per min×30 min, in saline) was initiated. Twenty five minutes into the infusion the rats were challenged again with TRAP (1 mg/kg, i.v.) and the changes in blood pressure were recorded.

[1]. Protease-activated receptors 1 and 4 mediate activation of human platelets by thrombin. J Clin Invest. 1999 Mar;103(6):879-87.

[2]. Rabbit and rat platelets do not respond to thrombin receptor peptides that activate human platelets. Blood. 1993 Jul 1;82(1):103-6.

[3]. Disparate effects of thrombin receptor activating peptide on platelets and peripheral vasculature in rats. Eur J Pharmacol. 1998 May 22;349(2-3):237-43.

Because of the role of thrombin and platelets in myocardial infarction and other pathological processes, identifying and blocking the receptors by which thrombin activates platelets has been an important goal. Three protease-activated receptors (PARs) for thrombin -- PAR1, PAR3, and PAR4 -- are now known. PAR1 functions in human platelets, and the recent observation that a PAR4-activating peptide activates human platelets suggests that PAR4 also acts in these cells. Whether PAR1 and PAR4 account for activation of human platelets by thrombin, or whether PAR3 or still other receptors contribute, is unknown. We have examined the roles of PAR1, PAR3, and PAR4 in platelets. PAR1 and PAR4 mRNA and protein were detected in human platelets. Activation of either receptor was sufficient to trigger platelet secretion and aggregation. Inhibition of PAR1 alone by antagonist, blocking antibody, or desensitization blocked platelet activation by 1 nM thrombin but only modestly attenuated platelet activation by 30 nM thrombin. Inhibition of PAR4 alone using a blocking antibody had little effect at either thrombin concentration. Strikingly, simultaneous inhibition of both PAR1 and PAR4 virtually ablated platelet secretion and aggregation, even at 30 nM thrombin. These observations suggest that PAR1 and PAR4 account for most, if not all, thrombin signaling in platelets and that antagonists that block these receptors might be useful antithrombotic agents.[1]

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Human platelets are aggregated and induced to release their granule contents and form thromboxane by peptides as short as 6-amino acid residues (SFLLRN) corresponding to the newly released N-terminus of the thrombin receptor that is cleaved by thrombin. Using washed platelets, we found that these responses to SFLLRN (2 to 6 mumol/L) were enhanced by fibrinogen. However, neither SFLLRN nor SFLLRNPNDKYEPF had any effect on washed rabbit or rat platelets, although they were fully responsive to human thrombin. Concentrations of the peptides as high as 100 mumol/L did not cause the platelets of rabbits or rats to change shape, aggregate, release granule contents, or form thromboxane. SFLLRN did not affect the extent of aggregation induced by adenosine diphosphate (ADP) or a low concentration of thrombin. Pig platelets responded to 50 mumol/L SFLLRN with reversible aggregation, which was enhanced by fibrinogen, but not accompanied by the release of dense granule contents. Guinea pig platelets aggregated and released granule contents in response to 25 or 50 mumol/L of SFLLRN, but responded with only shape change to lower concentrations. Thus, these experiments indicate that rabbit and rat platelets lack a functional response to human thrombin receptor peptides that fully activate the previously described human thrombin receptor, despite a full response of both rabbit and rat platelets to human thrombin, and that pig and guinea pig platelets have incomplete responses to these human thrombin receptor peptides. The results suggest that platelets of rabbits and rats, and perhaps guinea pigs and pigs, respond to thrombin through an alternative receptor that has also been suggested to be present on human platelets.[2]

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The hemodynamic and platelet effects of the thrombin receptor activating peptide SFLLRN (TRAP) were evaluated in rats. TRAP failed to aggregate rat platelets in vitro (platelet rich plasma) or in vivo in the pulmonary microcirculation. In contrast, TRAP aggregated washed human platelets. Intravenous injection of TRAP (1 mg/kg) in inactin-anesthetized rats produced a biphasic response in blood pressure characterized by an initial depressor response (-25 +/- 3 mmHg for 15-30 s) followed by a pronounced pressor response (50 +/- 7 mmHg for 2-3 min). This increase in blood pressure can be attributed to increases in total peripheral resistance since cardiac output remained unchanged. Further, only the pressor responses were observed in pithed rats suggesting a direct effect of TRAP in causing smooth muscle contraction. Consequently, rat platelets differ from human platelets in that they are resistant to TRAP whereas rat vasculature is highly sensitive to TRAP. These observations suggest that while the thrombin receptors on rat vasculature may be similar to those on human platelets, the receptors and/or the coupling mechanisms in rat platelets appear different from human platelets.[3]

C34H56N10O9

748.88

748.423

C, 54.53; H, 7.54; N, 18.70; O, 19.23

141136-83-6

9831933

Ser-Phe-Leu-Leu-Arg-Asn; H-Ser-Phe-Leu-Leu-Arg-Asn-OH

SFLLRN; H-SFLLRN-OH

White to off-white solid powder

1.834

11

11

24

53

1270

6

CC(C)C[C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CC(=O)N)C(=O)O)NC(=O)[C@H](CC1=CC=CC=C1)NC(=O)[C@H](CO)N

HAGOWCONESKMDW-FRSCJGFNSA-N

InChI=1S/C34H56N10O9/c1-18(2)13-23(30(49)40-22(11-8-12-39-34(37)38)29(48)44-26(33(52)53)16-27(36)46)42-31(50)24(14-19(3)4)43-32(51)25(41-28(47)21(35)17-45)15-20-9-6-5-7-10-20/h5-7,9-10,18-19,21-26,45H,8,11-17,35H2,1-4H3,(H2,36,46)(H,40,49)(H,41,47)(H,42,50)(H,43,51)(H,44,48)(H,52,53)(H4,37,38,39)/t21-,22-,23-,24-,25-,26-/m0/s1

L-seryl-L-phenylalanyl-L-leucyl-L-leucyl-L-arginyl-L-asparagine

TRAP 6; TRAP6; TRAP-6; Thrombin receptor activator peptide 6; Ser-Phe-Leu-Leu-Arg-Asn; 141136-83-6; L-Seryl-L-phenylalanyl-L-leucyl-L-leucyl-L-arginyl-L-asparagine; Ser-Phe-Leu-Leu-Arg-Asn; MFCD00238172; SFLLRN; (2S)-4-amino-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-amino-3-hydroxypropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]-4-methylpentanoyl]amino]-5-(diaminomethylideneamino)pentanoyl]amino]-4-oxobutanoic acid; SFLLRN; SFLLRN-OH

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, avoid exposure to moisture.

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

H2O : ~25 mg/mL (~33.38 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.)