nlm.nih.gov/protein/?term=txid8570[Organism:exp]), using the following parameters: peptide mass tolerance of ±0.1 Da, fragment mass tolerance of ±0.1 Da, oxidation as variable modifications in methionine and trypsin as enzyme. Amino acid analysis was performed on a Pico-Tag Analyzer (Waters Systems) as described by Henrikson and Meredith (1984). LmrTX PLA2, sample (30 μg) was hydrolyzed at 105 °C

for 24 h, in 6 M HCl (Pierce sequencing grade) containing 1% phenol Fulvestrant manufacturer (w/v). The hydrolysates were reacted with 20 μl of derivatization solution (ethanol:triethylamine:water:phenylisothiocyanate, 7:1:1:1, v/v) for 1 h at room temperature, after which the PTC-amino acids were identified and quantified by HPLC, by comparing their retention times and peak areas with those from a standard amino acid mixture. Modification of histidine residues was carried out as previously described by Diaz-Oreiro and Gutiérrez (1997). Briefly, approximately 3 mg of Lmr-TX were dissolved in 1 ml of 0.1 M Tris-HCI buffer, pH 8.0, containing 0.7 mM EDTA before adding 125 μl of p-bromophenacyl bromide (pBPB) solution (1.5 mg/ml in ethanol). The mixture was incubated for 24 h and the excess reagent was removed by ultrafiltration using an AMICON Ultra-15 centrifugal filter unit (3000 NMWL),

followed by lyophilization. 7–8 weeks old C57BL6 mice were anesthetized with xylazine (2%–16 mg/kg) and ketamine (10%–100 mg/kg) injected intramuscularly and placed in the supine position. Following a midline cervical Selleck GDC0199 incision,

the right common carotid artery was isolated and a Doppler Molecular motor flow probe (model 0.5 VB; Transonic Systems, Ithaca, NY) was applied. A 1.5-mW, 540-nm laser beam (Melles Griot, Carlsbad, CA) was applied to the artery from a distance of 6 cm. PLA2 was injected through lateral tail vein (3.75, 7.5 and 15 μg/animal) and after 10 min injury was initiated by injection into the lateral tail vein of rose bengal (50 mg/kg body weight; Fisher Scientific, Fair Lawn, NJ) dissolved in phosphate-buffered saline (PBS). Blood flow was monitored until complete and stable (5 min) occlusion occurred (Werneck et al., 2008). Citrated Mouse plasma was obtained after blood was withdrawn from cava vein in citrate 3.2% and centrifuged at 3000 rpm for 15 min at 25 °C. For APTT a 50 μl aliquot plasma was warmed to 37 °C for 2 min, 50 μl APTT reagent was added and after a 2 min incubation at 37 °C, 0.25 M CaCl2 was added and the clotting time was determined. For the PT test, 100 μl of CLOT PT reagent was incubated for 4 min at 37 °C and 50 μl of plasma was added, triggering the reaction. These analyses were performed in triplicate, using the APTT and PT Kit CLOT BIOS diagnosis (CLOT, Brazil) in a CLOTimer coagulometer. To assay APTT and PT ex vivo, C57BL6 mice were injected i.v. with LmrTX (15 μg/animal) and after 5, 15, 30, 60 and 90 min, blood was withdrawn and processed as described above. C57BL6 mice were injected i.v. with LmrTX (15 μg/animal).