S 2302

Full length activated FXIIa (α‐FXIIa) and commercial CTI were obtained from Enzyme Research Laboratories (Swansea, UK). S2302 (a chromogenic substrate peptide mimic) was obtained from Chromogenix (Epsom, UK). A codon‐optimized CTI cDNA was obtained from GenScript (Piscataway, NJ, USA). High‐purity‐grade (> 95%) synthetic peptides were obtained from GenScript. Purity was confirmed by reverse‐phase HPLC and mass spectrometry. DNA primers were obtained from Eurofins MWG (Ebersberg, Germany).

The catalytic activity of the native collinein-1 and rCollinein-mut was evaluated with the chromogenic substrate S-2302 by Chromogenix^® (Bedford, MA, USA) for plasma kallikrein (H-D-Pro-Phe-Arg-pNA•2HCl). Substrate hydrolysis was determined by incubating different doses of the proteins (1,25, 2,5, 5, and 10 μg) with 0.4 mM of substrates in Tris-HCl 0.05 M, pH 7.5, containing 0.05 M CaCl₂ for 40 min at 37 °C. Substrate hydrolysis was spectrophotometrically monitored at 405 nm using Versa Max Microplate reader (Molecular Devices, Sunnyvale, USA). Assays were performed in a series of three replicates, and the data were fitted with standard errors using GraphPad Prism software, version 5.0 (GraphPad Software, San Diego, USA). Statistical analysis of the results was performed using the Student’s t test or one-way ANOVA method with Tukey’s post-test, comparing all treatments to the negative controls and considering values of p < 0.05 as significant.

The following materials were obtained from the indicated sources: human fXIa, fIXa, fXa, thrombin, plasma kallikrein, and activated protein C (aPC) (Hematologic Technologies; Essex Junction, VT); recombinant tissue plasminogen activator (tPA) Alteplase® (Genentech; South San Francisco, CA); human α-fXIIa (Enzyme Research Laboratories; South Bend, IL); recombinant factor VIIa (fVIIa) Novoseven® (Novo Nordisk; Bagsværd, Denmark); Lys-plasmin and chromogenic substrates Spectrozyme FVIIa, Spectrozyme FIXa, Spectrozyme PCa, Spectrozyme tPA (Sekisui Diagnostics; Stamford, CT); S-2302, S-2366, S-2765 (Chromogenix; Milano, Italy); and Pefachrome TH 5244 (Pentapharm; Basel, Switzerland). CTI provided by Gamma (Pushchino, Russia) was extracted from corn, purified with ammonium sulfate, acetone precipitation, trypsin-affinity and ion-exchange chromatography, as described previously [17 (link)]. Luffa cylindrica trypsin inhibitor (LCTI-III) was chemically synthesized (Creative Dynamics; Shirley, NY). All other reagents were from (Sigma-Aldrich; St. Louis, MO) unless otherwise noted.

Lyophilized H. haemachatus crude venom was purchased from South African Venom Suppliers (Louis Trichardt, South Africa). Reagents for thromboplastin time, thrombin time and activated partial thromboplastin time (APTT) were from Helena Laboratories (Beaumont, Texas, USA). Reagents for N-terminal sequencing were from Applied Biosystems (Carlsbad, California, USA). The chromogenic substrates, S-2222, S-2288, S-2238, S-2251, S-2444, S-2366 and S-2302 were from Chromogenix (Milano, Italy). Spectrozyme FIXa was from American Diagnostica Inc (Stamford, Connecticut, USA). Superdex 30 HiLoad (16/60) column and Jupiter C₁₈ (5 μ, 300 Å, 4.6 × 250 mm) were purchased from GE Healthcare (Uppsala, Sweden) and Phenomenex (Torrance, California, USA), respectively. All other chemicals and reagents used were of the highest purity.

Serine proteases factor XIa (FXIa), factor IXa (FIXa), factor VIIa (FVIIa), factor X (FX), factor Xa (FXa) and Russell’s viper venom-X activator (RVV-X) were obtained from Haematologic Technologies Inc. (Vermont, USA), factor XIIa (FXIIa) was procured from Merck Calbiochem (Darmstadt, Germany), factor VIII (FVIII) from Creative Biomart (NY, USA), phospholipid blend from Avanti Polar Lipids Inc. (Alabama, USA) and tissue factor Innovin from Siemens (Murburg, Germany). The chromogenic substrates namely spectrozyme (MeSO₂-D-CHG-Gly-Arg-pNA.AcOH) was purchased from Sekisui Diagnostics (MA, USA), rest of the substrates like S-2366 (pyroGlu-Pro-Arg-pNA•HCl), S-2302 (H-D-Pro-Phe-Arg-pNA•2HCl), S-2222 (Bz-IIe-Glu(γ-OR)-Gly-Arg-pNA•HCl), S-2765 (Z-D-Arg-Gly-Arg-pNA•2HCl)S-2238 (H-D-Phe-Pip-Arg-pNA•2HCl) and S-2288 (H-D-Ile-Pro-Arg-pNA•2HCl) were procured from Chromogenix (NJ,USA).

Pooled diluted plasma (1:10 in 15 mM HEPES) was incubated with Pep19-2.5, and Dapptin was added for contact activation. In the positive control Dapptin (without peptid) and in the negative control water was added. 1 mM of the chromogenic substrate S-2302 (for PK/FXIIa activity, Chromogenix) or S-2366 (for FXI and Prot.C activity, Chromogenix) were added and the absorbance at 405 nm was measured at 37 °C over a period of 120 min. No endogenous proteolytic activity was measured in the absence of plasma.