Sample preparation: human α-thrombin and fibrinogen from human plasma were purchased from Haematologic Technologies (USA) and Sigma-Aldrich (United Kingdom), respectively. Oligonucleotides were suspended in 10 mM potassium phosphate buffer pH 7.4 and 100 mM KCl at a concentration of 10 μM. In order to induce folding, all oligonucleotide samples were annealed by heating to 90°C for 5 minutes and then slow cooling down in 50-60 minutes and storing at 20°C overnight.
Anticoagulant activity experiments: the α-thrombin-induced clotting of fibrinogen was measured spectrophotometrically. [66] A 1.8 mg mL -1 solution of fibrinogen in Phosphate Buffered Saline (PBS) was placed in a PS cuvette to which the oligonucleotide was added and left to equilibrate in the instrument for 5 min. Then, α-thrombin was added up to a final concentration of 5 nM. The time required for fibrin polymerization was determined from a UV scattering curve (380 nm), recorded over time in the presence of each oligonucleotide. Each curve was determined in triplicate at different oligonucleotide concentrations (20, 40, and 80 nM).
Clotting time was derived from the maximum of the second derivative of each scattering curve. The basal clotting time was determined in the absence of oligonucleotide (9 s). fibrinogen clotting times of NU172 and its variants were calculated by subtracting the basal clotting time.
Anticoagulant activity experiments: the α-thrombin-induced clotting of fibrinogen was measured spectrophotometrically. [66] A 1.8 mg mL -1 solution of fibrinogen in Phosphate Buffered Saline (PBS) was placed in a PS cuvette to which the oligonucleotide was added and left to equilibrate in the instrument for 5 min. Then, α-thrombin was added up to a final concentration of 5 nM. The time required for fibrin polymerization was determined from a UV scattering curve (380 nm), recorded over time in the presence of each oligonucleotide. Each curve was determined in triplicate at different oligonucleotide concentrations (20, 40, and 80 nM).
Clotting time was derived from the maximum of the second derivative of each scattering curve. The basal clotting time was determined in the absence of oligonucleotide (9 s). fibrinogen clotting times of NU172 and its variants were calculated by subtracting the basal clotting time.