These experiments were performed by specialists at the School of Chemistry’s NMR Unit, University of Edinburgh (Edinburgh, Scotland, UK). The sample was dissolved in 350 μl D2O (Sigma-Aldrich) and placed into a Shigemi tube. Spectra were acquired using 800 or 400 MHz NMR spectrometers (Bruker Daltonics, Bremen, Germany). 1D 1H spectrum was measured using 64 scans, acquisition time of 4.1 s, relaxation time of 5 s, and flip angle of 30°. A 2D Correlation Spectroscopy (COSY) spectrum was acquired

using t1 and t2 acquisition times of 148 and 256 ms, 2 scans per increment and a relaxation time of 2 s resulting in the total acquisition Ponatinib supplier time of 2 h 40 min. The 2D Total Correlation Spectroscopy (TOCSY) spectrum was acquired in 1 h, using t1 and t2 acquisition times of 37 and 256 ms, 4 scans per increment and a DIPSI-2 mixing time of 150 ms. The 2D Nuclear Overhauser Effect Spectroscopy

(NOESY) spectrum was acquired in 3.5 h, using t1 and t2 acquisition times of 37 and 256 ms, 8 scans per increment and a mixing time of 400 ms. The 2D 1H-13C Heteronuclear Single Quantum Coherence (HSQC) spectrum was acquired in 2 h, using t1 and t2 acquisition times of 30 and 128 ms, 12 scans per increment. The 1D 13C NMR spectrum Talazoparib was acquired in 6.5 h using 8k scans, the acquisition time of 340 ms and the relaxation time of 2.5 s. The 400 MHz 1D 1H spectra with and without 31P decoupling were acquired using parameters similar to those used for the 800 MHz 1D 1H spectrum. 31P NMR spectra were acquired in 20 min using 512 scans, acquisition time of 0.5 s and a relaxation ifoxetine time of 2 s. The 2D 1H-31P HMBC spectra were acquired in magnitude mode using a phase cycled Heteronuclear Multiple Bond Coherence (HMBC) pulse sequence optimized for

1H-31P coupling constant of 10 Hz. The spectra were acquired in 3.52 h using t1 and t2 acquisition times of 20 and 250 ms; 32 scans per increment were accumulated. The sample was analysed at pH 3.8 and 6.5, by adjusting the pH of the sample (3.8) to 6.5 after titration. In order to evaluate the relevance of ADP to the vasoactive effect of the venom as a whole, concentration-response curves were performed in rat aortic rings, in the absence or presence of suramin, a P2-purinergic receptor antagonist. Increasing cumulative concentrations of Lasiodora sp. venom (0.06-64 μg/ml) or ADP (0.001-316 μM) were added in aortic rings with functional endothelium pre-contracted with phenylephrine (0.1 μM). The experiments were repeated in the presence of suramin (100 μM), added to the bath 20 min prior to the addition of phenylephrine. Results are expressed as means ± standard error of the mean (S.E.M.). Results from contractile experiments were expressed as percentage decrease in the maximal contraction induced by phenylephrine, and the point when the basal line was reached was considered 100% relaxation.