Cyproheptadine

Serotonin hydrochloride, pizotifen and ADP were obtained from Sigma Aldrich (St. Louis, MO), cyproheptadine and EMD 281014 were obtained from Tocris Bioscience (Bristol, UK), clopidogrel was purchased from LKT Laboratories, Inc. (St. Paul, MN), stir bars and other disposables were from Chrono-Log (Havertown, PA), and U46619 was obtained from Cayman Chemical (Ann Arbor, MI). Src antibody, FITC-conjugated Annexin V, anti–P-selectin, and PAC-1 antibodies were purchased from Cell Signaling Technology, Inc. (Danvers, MA). The anti-phosphotyrosine antibody was from BD Biosciences, (Franklin Lakes, NJ). Fura-2 acetoxymethyl ester (fura-2/AM) and Pluronic® F-127 were from Invitrogen (Grand Island, NY). The C57BL/6 mice were obtained from Jackson laboratory (Bar Harbor, ME). Platelet count was determined using an automated hematology analyzer (Drew Scientific Dallas, TX).

The following drugs were administered: ATP (100 μM), methysergide (100 μM), WAY-100635 (1 μM), L-NAME (100 μM), L-NMMA (100 μM), atropine (1 μM) all from Sigma (Deisenhofen, Germany) cyproheptadine (5 μg/ml) (TOCRIS Bioscience, Bristol, UK).
Application of drugs taken from a stock solution (see above) was performed with a pipette into the organ bath solution; end concentration was achieved by gently mixing the buffer with a pipette. All preparation and experiments were carried out in HEPES solution consisting of : 20 mM HEPES, 4,5 mM KCl, 2,5 mM CaCl₂, 11 mM Glucose, 140 mM NaCl, 1 mM MgCl₂, pH was adjusted to 7.4 at 30°C or 4–8°C for tissue preparation using NaOH (4 molar).
TNF-α (PeproTech, Rocky Hill, New Jersey) was taken out of a stock solution (100 ng/μl, diluted in water) that was freshly prepared for each experimental day.

KYNA was purchased from Sigma-Aldrich Ltd. (Budapest, Hungary). The following receptor blockers were applied: cyproheptadine, a nonselective 5-HT2 serotonergic receptor antagonist, in a dose of 5 mg/kg (Tocris, Bristol, UK); phenoxybenzamine hydrochloride, a nonselective α-adrenergic receptor antagonist, in a dose of 2 mg/kg (Smith Kline and French, Hertz, UK); naloxone, a nonselective opioid receptor antagonist, in a dose of 0.3 mg/kg (Endo Lab Inc., Malvern, PA, USA), haloperidol, a D2, D3, D4 dopamine receptor antagonist, in a dose of 10 μg/kg (Richter Gedeon Plc., Budapest, Hungary), propranolol hydrochloride, a nonselective β-adrenergic receptor antagonist, in a dose of 2 mg/kg (ICI Ltd., Macclesfield, UK), atropine sulfate, the nonselective muscarinic acetylcholine receptor antagonist in a dose of 2 mg/kg (EGIS, Budapest, Hungary). The effective doses of the receptor antagonists have been determined based on the previous studies published and our previous work. The doses are calibrated in which no change in tested behaviors is observable [68 (link),69 (link),70 (link)]. KYNA was freshly dissolved in 0.9% aqueous saline solution and its pH was set to approximately 7.4 before use. The control animals received only 0.9% saline solution.