Table 1.
Semi-quantitative determination of procoagulant activity of venom of 20 snake species.
The extent of pro- and anti-coagulant activity is graded from strong (+++) to weak (+), where 0 represents no activity detected. Bold species names indicated those venoms selected for in depth characterisation.
Table 2.
Assignment of coagulopathic venom toxins based on masses detected for intact bioactive components and Mascot hits after tryptic digestion.
Fig 1.
Detection of coagulation interfering compounds of Bothrops asper by correlating MS and bioassay data obtained upon LC analysis of crude snake venom (5 mg/mL).
i: UV chromatograms detected at 220 and 254 nm. ii: Bioactivity chromatograms obtained after fractionation by plotting processed results of the plasma coagulation assay against time (data was acquired for 80 cycles (±120 min); the positive peaks (left) and negative peaks (right) indicate the presence of procoagulant and anticoagulant bioactive compounds, respectively. iii: MS chromatograms (TIC), iv: Extracted-ion chromatograms (XICs) of m/z-values corresponding to bioactive peaks. For further experimental conditions, see Materials and Methods section.
Fig 2.
Identification of coagulopathic compounds from Bothrops jararaca (A), Calloselasma rhodostoma (B), Daboia russelii (C), Echis ocellatus (D), Dispholidus typus (E) and Oxyuranus scutellatus (F)by correlating MS data with bioassay data. i: UV trace of the snake venoms at 220 and 254 nm obtained by LC-MS. ii: bioactivity chromatograms obtained by plotting the results of the plasma coagulation assay in Prism software. The peaks with positive maxima (right) and negative (left) minima indicate the presence of procoagulant and anticoagulant bioactive compounds respectively (Data was acquired for 80 cycles (±120 min). 6-s resolution fractions were collected onto 384 well plates by the nanofractionator after 50-μL injection of crude snake venom at a concentration of 5 mg/mL. iii: LC-MS chromatograms displaying the total ion current (TIC), iv: the extracted ion currents (XICs) of the m/z-values corresponding to bioactive peaks found in the MS spectra.
Fig 3.
The molecular weight distribution of the various anti- and pro-coagulant toxins identified in the venom of each of the seven snake species.
The number and molecular weight range of toxins identified from each snake species are summarized for procoagulant (A) and anticoagulant (B) bioactive compounds. Toxin identifications are derived from those described in Table 2 and their respective molecular weights were calculated by drawing structures in chemdraw and adding appropriate PTMs. The full data can be found in detail in the S2 Table. C) Molecular weight comparisons of the anticoagulant and procoagulant toxins detected from all seven snake venoms. Boxes show the interquartile range of each dataset, with the bold horizontal line representing the median value, and each data point is illustrated by a dot. Statistical comparisons, via non-parametric factorial analysis, reveals that procoagulant venom toxins exhibit significantly higher molecular weights than anticoagulants (P = 2.22e-16, F = 163.52).
Fig 4.
Identification of the various anti- and pro-coagulant toxins associated with the areas in the bioactivity profiles where pro- and anti-coagulant activities were observed.
Panel A shows all the toxins detected where pro and anticoagulant activity was observed. This panel is constructed with the S1 Table displayed in the Supplementary information. Panel B shows toxins that could be matched to observed bioactivity with high confidence due to correlation with MS data or abundance of the toxin and its known activity. This panel is constructed with the data displayed in Table 2.