Ascent software v2

For each of the methods performed on the Fluoroskan Ascent™ (Thermo Scientific, Vantaa, Finland) Microplate Fluorometer (Cat#1506450, Thermo Scientific, Vantaa, Finland), data was collected using Ascent^® Software v2.6 (Thermo Scientific, Vantaa, Finland). All raw data were firstly blank corrected using the Ascent^® software (v2.6, Thermo Scientific, Vantaa, Finland) package and then exported for further analysis using Windows Excel 2016 and GraphPad PRISM 7.0 (GraphPad Prism Inc., La Jolla, CA, USA).
Graphs: Using Excel, averages of blank corrected data triplicates were calculated. From these averages, maximum absorbance was calculated and absorbance value plotted in a column graph for relative percentages.
Two-way (RM) ANOVA and Tukey’s Post-hoc test: the blank corrected data was transformed, setting the top value as 100 and lowest as 0 across sub columns within a data set. A two-way repeated measures (RM) ANOVA was performed, followed by a Tukey’s post-hoc multiple comparison test, comparing every mean with every other mean.

Activation of human Factor X (FX) and prothrombin by the venoms were measured with a Fluoroskan Ascent™ Microplate Fluorometer with Ascent^® Software v2.6 (Thermo Fisher Scientific, Vantaa, Finland) using the following protocol: 10 µL venom (0.01, 0.05, and 0.1 µg/μL) in dilution buffer (150 mM NaCl, 50 mM Tris-HCl, pH 7.4) was plated in at least triplicate on a 384-well plate along with 10 µL phospholipid (Stago Cat#00597 in O.K. Buffer) and 10 µL of human recombinant FX or prothrombin (0.01 μg/mL) (Haematologic Technologies, VT, USA) in dilution buffer. Venom controls substituted 10 μL coagulation factor with 10 μL dilution buffer. Vehicle controls substituted 10 μL venom and 10 μL coagulation factor with 20 μL dilution buffer. Positive controls substituted 10 μL venom with dilution buffer and 10 μL coagulation factor with 10 µL (0.01 μg/mL) of human recombinant FXa or thrombin (Haematologic Technologies, Inc.). Plates were loaded into the fluorometer (pre-incubated to 37 °C), and 70 µL quenched substrate was dispensed into each well (1 µL substrate in 500 μL enzyme buffer: 150 mM NaCl, 50 mM Tris-HCl, 5 mM CaCl, pH 7.4. Fluorogenic Peptide Substrate Cat#ES002 used for FX activation assay; Cat#ES011 for prothrombin). Fluorescence was monitored (excitation/emission: FX–390 nm/460 nm, prothrombin–390 nm/460 nm) for 300 cycles over ~150 min.

A working stock solution of freeze dried venom was reconstituted in a buffer containing 50% MilliQ/50% glycerol (>99.9%, Sigma, St. Louis, MO, USA) at a 1:1 ratio to preserve enzymatic activity and reduce enzyme degradation. Varying concentrations of crude venom (10 ng/µL and 50 ng/µL) were plated out in triplicates on a 384-well plate (black, Lot#1171125, nunc™ Thermo Scientific, Rochester, NY, USA) and measured by adding 90 µL quenched fluorescent substrate per well (total volume 100 µL/well, 10 µL/5mL enzyme buffer, 150 mM NaCl, and 50 mM Tri-HCl (pH 7.3), Fluorogenic Peptide Substrate, R&D systems, Cat#ES001 & ES011, Minneapolis, MI, USA). Fluorescence was monitored by a Fluoroskan Ascent™ (Thermo Scientific, Vantaa, Finland) Microplate Fluorometer (Cat#1506450, Thermo Scientific, Vantaa, Finland) (Cat#ES001 for Matrix Metalloprotease at an excitation of 320 nm, emission at 405 nm; Cat#ES011 for Kallikrein at an excitation of 390 nm, emission at 460 nm) over 400 mins or until activity had ceased. Data was collected using Ascent^® Software v2.6 (Thermo Scientific, Vantaa, Finland).