Human plasma fibrinogen

Human plasma αT (EC 3.4.21.5) and ProT were purchased from Haematologic Technologies (Essex Junction, VT, USA). Ecarin from Echis carinatus venom, bovine pancreatic trypsin, human plasma fibrinogen, Ac-Tyr-NH₂, Ac-Phe-NH₂, fluorescein isothiocyanate, and PABA were purchased from Sigma (St. Louis, MO, USA). The chromogenic substrate S2238 was from Chromogenix (Milan, Italy). SPAN12 monoclonal antibody was purchased from Beckman Coulter (Brea, CA, USA). Hirugen (⁵⁴GDFEEIPEEY*LQ⁶⁵) and [F]-hirugen^{58 (link)}, fibrinogen γʹ–peptide (⁴⁰⁸VRPEHPAETEY*DSLY*PEDDL⁴²⁷)^{63 (link)}, PAR1(38–60) (³⁸LDPR↓SFLLRNPNDKYEPFWEDDE⁶⁰)^{57 (link)}, Hir(1–47)^{51 (link)}, and αSyn(103–140) were synthesized by standard solid phase N^α-fluorenylmethyloxycarbonyl chemistry on a PS3 automated synthesizer (Protein Technologies Int., Tucson, AZ, USA), purified to homogeneity (> 98%) by semipreparative RP-HPLC, and characterized by high resolution mass spectrometry. Notably, Y* indicates phosphorylated Tyr residues. N^α-Fmoc-protected amino acids, solvents, and reagents for peptide synthesis were purchased from Applied Biosystems (Forster City, CA, USA) or Bachem AG (Bubendorf, Switzerland). Salts, solvents, and other reagents were of analytical grade and purchased from Sigma or Fluka (Darmstadt, Germany).

To assess the fibrinogenolytic activity of the immunogens and their inhibition by the resulting experimental antivenoms, we used an SDS-PAGE gel electrophoresis approach using human plasma fibrinogen (Sigma-Aldrich, UK). The recombinant toxin immunogens (3 μg, 1 mg/mL) and various crude venoms (1.05 μg, 0.3 mg/mL) were incubated with human fibrinogen (3.75 µg, 2.5 mg/mL, Sigma-Aldrich, UK) for two hours at 37 °C. All immunogen samples were also used in neutralisation experiments following a pre-incubation step at 37 °C for 15 min with 1.75 µg (1 mg/mL) of the corresponding toxin-specific experimental antivenom (i.e., for ancrod and C. rhodostoma venom, anti-ancrod was used). Next, ten well 8% SDS-PAGE gels were hand-cast using the following approach: 10 mL resolving gel (4.7 mL H₂O, 2.5 mL 1.5 M Tris pH 8.8, 2.7 mL 30% bis-acrylamide, 50 µL 20% SDS, 100 µL 10% APS and 7 µL TEMED); 4 mL of 4% stacking gel (2.7 mL H₂O, 0.5 mL 1M Tris pH 6.8, 800 µL 30% bis-acrylamide, 20 µL 20% SDS, 40 µL 10% APS and 4 µL TEMED). Thereafter, 10 µL of each sample was loaded on the gel and analysed under reducing conditions as described earlier. The negative control consisted of human fibrinogen only (3 µg, 2.5 mg/mL, Sigma-Aldrich, UK).

E. coli O111:B4 LPS and human plasma fibrinogen were purchased from Sigma-Aldrich. Samples were reconstituted in warm phosphate buffered saline and incubated for 1 h at 37°C with shaking. LPS was then sonicated for 1 h at 60°C. Fibrinogen solutions were passed through a 0.2 µm polyethersulfone syringe filter and concentrations were determined by UV absorbance (E_1% = 15.1 at 280 nm). Samples were then diluted with buffer to the required concentration and degassed. ITC experiments were performed at 37°C on a MicroCal Auto-iTC200 system (GE Healthcare) in high-gain mode at a reference power of 10 µcal s⁻¹, with an initial 0.5 µl (1 s) injection followed by 15 2.5 µl (5 s) injections with 300 s spacing. For longer titrations, the syringe was refilled and injections continued into the same cell sample. Control runs were performed in which cell samples were titrated with buffer and syringe samples were titrated into buffer, and data from these runs were subtracted from the experimental data as appropriate. Data analysis was performed in Origin, using the supplied software (MicroCal).