S 2444

FSAP-SPD was incubated with pro-uPA (Grünenthal, Stolberg, Germany) and its activation was measured by adding 0.2 mM of the chromogenic substrate S-2444 (L-pyroglutamyl-glycyl-L-arginine-p-nitroanilinedihydro-chloride) (Chromogenix). Absorbance was followed for 60 min at 37 °C at 405 nm in a microplate reader. FVIIa clotting activity was determined in the presence of a recombinant soluble mutant tissue factor (TF)^{23 (link)}. Briefly, FVIIa solutions were mixed with equal volumes of FVII-deficient plasma (Roche Diagnostics, Mannheim, Germany), a mixture of TF and phospholipids (PTT reagent, Diagnostica Stago, Asnieres, France) and 25 mM CaCl₂. Clotting and turbidity changes were monitored at 37 °C and 405 nm in a microplate reader. In each well the final concentration of total FVII/FVIIa was set to 5 nM and TF to 60 nM. The level of FVIIa generated was quantified relative to the WHO 1^st International Standard FVIIa concentrate (1 IU/ml FVIIa corresponds to 20 ng/ml or 0.5 nM).

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.

Aliquots of samples were normalized with respect to protein concentration (A280 nm = 0.1) and analyzed for amidolytic activity on Glu-Gly-Arg-pNA (S-2444; Chromogenix, Mölndal, Sweden) as described previously [20 (link)]. Activity of uPA (10 ng/mL) in solution in 50 mM Tris (tris(hydroxymethyl)aminomethane)-HCl, 100 mM NaCl, pH 7.4, at 37 °C was studied by continuous assay using S-2444. uPA and S-2444 were equilibrated at 37 °C in the spectrophotometer sample cuvette. The absorbance at 406 nm was monitored over time. Absorbance measurements were transformed using the first derivative function to yield S-2444 (0.3 mM) hydrolysis velocities (ΔA406/Δt).