3 amp

For a list of abbreviations, see Supplementary Table 1. Nucleobases A and T, nucleosides Ado, Guo and Urd, nucleotides (2′-AMP, 3′-AMP, 5′-AMP, 2′,3′-AMP, 3′,5′-AMP, 5′-CMP, 2′,3′-CMP, 5′-GMP, 3′,5′-GMP and 5′-UMP), deoxyribonucleotides (dAMP, dCMP, dGMP and dTMP), 2-aminoazoles (2AO, 2AI and 2AT), oligomers of Gly (Gly, GlyGly and GlyGlyGly) and TMP were purchased from Sigma-Aldrich (USA). Nucleobase C was purchased from Carl Roth GmbH (Germany) and nucleobases U and G and nucleosides L-Cyd and D-Cyd were purchased from Biosynth Carbosynth (UK). Nucleotides 2′-CMP, 3′-CMP, 3′,5′-CMP, 2′,3′-GMP, 2′,3′-UMP and 3′,5′-UMP were purchased from Biolog (USA). For the proteogenic amino acids, an Amino Acid Mixture was used (L4461, Promega, USA) and compared with individual amino acids (Sigma-Aldrich, USA). For experiments on non-proteogenic amino acids, a mixture of amino acids and small molecules was used (A9906, Sigma-Aldrich, USA).

Thermodynamic binding parameters of CxD7L1 and CxD7L2 to several pro-hemostatic ligands were tested using a Microcal VP-ITC microcalorimeter. The panel of substances tested included several nucleosides/nucleotides or derivates (ATP, ADP, 5′-AMP, 3′-AMP, cyclic AMP, adenosine, GTP, TTP, inosine, sodium polyphosphate, Sigma-Aldrich), biogenic amines (epinephrine, norepinephrine, histamine, serotonin, Sigma-Aldrich), and pro-inflammatory/pro-hemostatic lipid compounds (LTB₄, LTC₄, LTD₄, LTE₄, arachidonic acid, and the stable analog of TXA₂: U-46619, Cayman Chemicals). Ligands and protein solutions were prepared in 20 mM Tris–HCl, pH 7.4, 150 mM NaCl (TBS) at 30 and 3 µM, respectively. Lipid ligands were prepared by evaporating the ethanol or chloroform solvent to dryness under a stream of nitrogen. Lipid ligands were further dissolved in TBS and sonicated for 10 min (Branson 1510) to ensure dissolution. Lipid ligands were used at 50 µM of ligand and 5 µM of protein. Injections of 10 µL of ligand were added to the protein samples contained in the calorimeter cell at 300 s intervals. Experiments were run at 30 °C. Thermodynamic parameters were obtained by fitting the data to a single-site binding model in the Microcal Origin software package version 7 (OriginLab). For saturation studies, CxD7L2 protein was pre-incubated with 50 µM serotonin for 30 min and titrated with LTD₄.

Ectonucleotidase activity was determined by the rate of inorganic phosphate (Pi) released, as previously described (Rodrigues et al., 2015 (link)). Briefly, intact cells (3 x 10⁹ cells) were incubated for 1 h at room temperature (RT) in 0.5 ml of reaction mixture containing 116 mM NaCl, 5.4 mM glucose, 50 mM HEPES-MES-Tris buffer (pH 4.0), and 5 mM of 5′AMP (Sigma-Aldrich) or 5 mM of 3′AMP (Sigma-Aldrich) as substrates. Additional tubes with substrates incubated with the ectonucleotidase inhibitor ammonium tetrathiomolybdate (TTM; 100 µM) were also prepared. The reaction was stopped by the addition of 1 ml of 25% charcoal in 0.1 M HCl. Then, the mixture was centrifuged and 0.1 ml of the supernatant was added to 0.1 ml of Fiske Subbarow reactive mixture. The absorbance of the released Pi was measured spectrophotometrically at 650 nm. Ecto-3′ and ecto-5′-nucleotidase activities were calculated by subtracting the nonspecific 3′AMP and 5′AMP hydrolysis in blanks; the concentration of Pi released in the reaction was determined using a standard curve of Pi.