PAR1/rotease activating receptor 1 (IC50 = 96 nM)
Protease-activated receptor 1 (PAR1) [1]

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].

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In Xenopus oocytes heterologously expressing human PAR1, the peptide SFLLRN triggered calcium mobilization with an EC50 roughly two orders of magnitude lower than that of GYPGQV for PAR4 activation. SFLLRN showed no activity at PAR4. [1]
- In human platelets, SFLLRN (10 μM) induced platelet aggregation measured as change in light transmission. [1]
- Preincubation of platelets with SFLLRN (100 μM) in the presence of prostaglandin E1 at room temperature for 30 minutes without stirring rendered platelets refractory to subsequent stimulation by SFLLRN (500 μM) but did not affect responsiveness to the PAR4-activating peptide GYPGKF (500 μM). [1]
- Desensitization of PAR1 with SFLLRN markedly inhibited platelet aggregation in response to 1 nM thrombin, but was largely ineffective against 30 nM thrombin (only slowing aggregation slightly). [1]

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

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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.

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Platelet aggregation and secretion assay: Washed human platelets were used. For desensitization studies, platelets resuspended from the first platelet pellet were incubated with SFLLRN (100 μM) in the presence of prostaglandin E1 at room temperature for 30 minutes without stirring, then washed by centrifugation. Aggregation was measured as increase in light transmission after exposure to agonists. Secretion was measured as ATP release using lumiaggregometry. [1]
- Xenopus oocyte functional assay: Oocytes were microinjected with 25 ng of PAR1 cDNA per oocyte. Peptide-triggered 45Ca release was measured. Data were expressed as fold increase over baseline. Surface expression of PAR1 was measured with anti-FLAG monoclonal antibody. [1]

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.

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[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.

Since thrombin and platelets play crucial roles in myocardial infarction and other pathological processes, identifying and blocking thrombin-activated platelet receptors has been an important research goal. Currently, three protease-activating receptors (PARs) for thrombin are known: PAR1, PAR3, and PAR4. PAR1 functions in human platelets, and a recently discovered PAR4-activating peptide can activate human platelets, suggesting that PAR4 may also play a role in these cells. Whether PAR1 and PAR4 are the main mechanisms by which thrombin activates human platelets, or whether PAR3 or other receptors are also involved, remains unclear. We investigated the roles of PAR1, PAR3, and PAR4 in platelets. PAR1 and PAR4 mRNA and protein expression were detected in human platelets. Activation of any one receptor was sufficient to trigger platelet secretion and aggregation. Inhibition of PAR1 alone, using antagonists, blocking antibodies, or desensitization therapy blocked 1 nM thrombin-induced platelet activation, but had only a slight inhibitory effect on 30 nM thrombin-induced platelet activation. Inhibition of PAR4 alone with blocking antibodies had no significant effect at either thrombin concentration. Notably, simultaneous inhibition of PAR1 and PAR4 almost completely suppressed platelet secretion and aggregation, even at a thrombin concentration of 30 nM. These observations suggest that PAR1 and PAR4 mediate most (if not all) of thrombin signaling in platelets, and that antagonists blocking these receptors may be an effective antithrombotic agent. [1]

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Human platelets aggregate under the influence of peptides as short as 6 amino acid residues (SFLLRN) and are induced to release their particulate contents to form thromboxane. These peptides correspond to the N-terminus of newly released thrombin receptors after thrombin cleavage. Using washed platelets, we found that fibrinogen could enhance these responses induced by SFLLRN (2 to 6 μmol/L). However, SFLLRN and SFLLRNPNDKYEPF had no effect on washed rabbit or rat platelets, although they were completely sensitive to human thrombin. Peptides at concentrations up to 100 μmol/L did not induce changes in platelet shape, aggregation, release of granular contents, or thromboxane formation in rabbit or rat platelets. SFLLRN did not affect the degree of platelet aggregation induced by adenosine diphosphate (ADP) or low concentrations of thrombin. Porcine platelets showed reversible aggregation in response to 50 μmol/L SFLLRN, which was enhanced by fibrinogen, but without the release of dense granular contents. Guinea pig platelets showed aggregation and release of granular contents in response to 25 or 50 μmol/L SFLLRN, but only shape changes in response to lower concentrations of SFLLRN. Therefore, these experiments demonstrate that although rabbit and rat platelets fully respond to human thrombin, they lack a functional response to thrombin receptor peptides that fully activate the previously described human thrombin receptor; and porcine and guinea pig platelets also do not fully respond to these human thrombin receptor peptides. The results suggest that platelets in rabbits and mice (and perhaps guinea pigs and pigs) respond to thrombin via another receptor, which is also thought to be present on human platelets. [2]

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The hemodynamics and platelet effects of the thrombin receptor activating peptide SFLLRN (TRAP) were evaluated in mice. 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. In rats anesthetized with sodium thiopental, intravenous administration of TRAP (1 mg/kg) elicited a biphasic response in blood pressure, characterized by an initial depressor response (-25 ± 3 mmHg, lasting 15–30 seconds) followed by a significant pressor response (50 ± 7 mmHg, lasting 2–3 minutes). The increase in blood pressure was attributable to an increase in total peripheral resistance, as cardiac output remained constant. Furthermore, only a pressor response was observed in rats with spinal cord transection, suggesting that TRAP directly induces smooth muscle contraction. Therefore, the difference between rat platelets and human platelets is that rat platelets are resistant to TRAP, while the rat vascular system is highly sensitive to TRAP. These observations suggest that although the thrombin receptors on the rat vascular system may be similar to those on human platelets, the receptors and/or coupling mechanisms in rat platelets appear to be different from those in human platelets. [3]

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SFLLRN is a synthetic peptide that mimics the first six amino acids of the new NH2-terminus unmasked by thrombin cleavage of PAR1. It functions as a PAR1 agonist and activates the receptor independent of proteolysis. [1]
- Activation of PAR1 by SFLLRN is sufficient to trigger human platelet secretion and aggregation. [1]
- SFLLRN has been used as a pharmacological probe of PAR function in various cell types. [1]
- Iterations around PAR1's tethered ligand sequence SFLLRN have led to the development of potent peptide-based antagonists (e.g., BMS200261) that block human platelet activation by SFLLRN itself and by low concentrations of thrombin. [1]

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.)