Quantifying Plasma Amidase Activity

Plasma samples were prepared from citrate-blood collections, centrifuged to prepare the platelet-free plasma and immediately frozen at −80°C. The spontaneous amidase activity was evaluated using the peptide substrate HD-Pro-Phe-Arg-pNA (1 mM; Bachem), representing the P1-P′1 scissile bond by kallikrein at the C-terminus of BK. This assay refers to enzymes with spontaneous amidase activity, i.e. the Serine proteases of contact phase and fibrinolysis (kallikrein, FXII, plasmin and tissue-type plasminogen activator). Spontaneous amidase activity was kinetically monitored by the A₄₀₅ at 30°C (ThermoFisher Spectrophotometer), and expressed in nmol⋅min⁻¹⋅mL⁻¹ (molar extinction coefficient of p-nitroaniline 8800 M⁻¹⋅cm⁻¹).
In order to refer to plasma proenzyme activation, the contact system was activated by cold pre-incubation of plasma sample with dextran sulfate (12.5 mg⋅mL⁻¹) [18] (link), then the subsequent enzyme activity was assessed using the peptide substrate HD-Pro-Phe-Arg-pNA and monitored by the A₄₀₅ at 30°C.