SVMP classification and abundance in Echis venom.

(a) Pie charts displaying Echis ocellatus and Echis carinatus sochureki venom composition, with SVMP coloured in cyan (1). (b) Simplified schematic of mature PI, PII and PIII SVMP architecture, showing metalloproteinase domain (MP, cyan), disintegrin domain (Dis, green) and cysteine-rich domain (C-rich, magenta). (c) AlphaFold 3 predicted structural models of PI and PII SVMPs from Echis ocellatus, and PIII SVMP from Echis carinatus sochureki. Domain names and colour coding as in panel b. Zn2+ ions (orange) in the active site are shown as spheres.

PI, PII and PIII SVMP zymogen expression.

(a) Above: Schematic of PI SVMP zymogen with N-terminal prodomain (Pro, salmon), propeptide (purple). Below: left: two views of AlphaFold 3 prediction of PI zymogen model; right: zoom in on active site Zn2+ ion coordinated by histidines and propeptide cysteine. Expression of SVMP zymogens monitored by (b) cell viability, (c) YFP fluorescence (PIII harvested on day 4), (d) Western blot analysis using HRP-conjugated anti-Penta-His antibody. SNP: supernatant+pellet, SN: supernatant, M: media (10x concentrated). Grey bars: non-toxic protein expression control, cyan: PI SVMP, green: PII SVMP, magenta: PIII SVMP. SVMP zymogen expression was repeated five times.

SEC purification of SVMP zymogens and auto-activation.

Size exclusion chromatograms and SDS-PAGE of (a) PI zymogen (PIzymΔC lacks C-terminal tags), (b) PII zymogen, and (c) PIII zymogen after IMAC and IEX. PIIIzym partial cleavage separates the prodomain (Pro) and the mature PIII. Elution volumes of MW calibration markers are indicated in the chromatograms as black arrows. (d) Activation of PI zymogen into metalloproteinase and prodomain. C (control): no 18-hour incubation. (e) Activation of PII zymogen into metalloproteinase-disintegrin and disintegrin domain (prodomain cannot be definitively identified). After 1 week only 8 kDa and 23 kDa bands remain. (f) Activation of PIII zymogen into prodomain and mature PIII. At higher Zn2+ concentrations, the prodomain is degraded. C (control): no 18-hour incubation. SVMP zymogen purifications and activations were repeated five times.

In vitro SVMP activity and substrate specificity.

Casein degradation in the presence of increasing amounts of (a) PI, (b) PII and (c) PIII SVMPs. Orange arrowheads: domains αS1, αS2, β and κ. Fibrinogen degradation in the presence of increasing amounts of (d) PI, (e) PII and (f) PIII. Orange arrowheads: fibrinogen chains Aα, Bβ and γ. C (control): incubation in the presence of 5 mM EDTA. (g) PIII SVMP (20 nM) activity towards the fluorogenic peptide substrate ES010. (h,i) Degradation of insulin B by (h) PIII and (i) recombinant (blue) and Echis ocellatus PI (red). Full-length insulin (FL) and degradation products (*) are marked above the peaks. All degradation assays were repeated three times. The fluorogenic peptide assay shows the result of three independent repeats.

SVMP blood coagulation, platelet aggregation and cytotoxicity.

(a) Inhibition of platelet aggregation by PI, PII (mature and zymogen) and PIII. Control: PRP with buffer. Data are presented as the mean of % aggregation ± SEM. A two-way ANOVA was conducted, followed by simple effects analysis (Dunnett post-test) to compare column means (SVMPs) within each row (concentrations). ****: p ≤ 0.0001, **: p ≤ 0.01. The assay was carried out in experimental duplicate and biological triplicate. (b)-(d) Thrombin clot time assays using the ACL TOP coagulation analyser. Citrated human blood was spiked with (b) thrombin, (c) a pool of native Echis ocellatus PIII SVMPs, and (d) the recombinant PIII SVMP. Change in absorbance (blue) is plotted on the left Y axis, rate of change (1st derivative: orange; 2nd derivative: red) is plotted on the right Y axis. Assays were repeated three times. (e) Coagulation profiles of the recombinant SVMPs and Echis ocellatus venom in bovine plasma over 30 minutes at an absorbance of 595 nm. Data points represent the mean of three individual values ± SD. The experiment was repeated two times. (f) MTT cytotoxicity assay showing viability compared to the buffer-only control of HaCaT cells after 24-hour incubation with activated PI, PII and PIII SVMPs. Bars show the results of two independent repeats.