Detection of PC in human plasma

PC determination was performed according to manufacturer’s instructions (Biophen Protein C, Aniara Diagnostica, OH) with minor modifications. Briefly, 25 μl of human control normal plasma (Biophen normal control plasma ref A223201) diluted 1:1 with sterile saline solution (0.9% NaCl) were incubated with 50 μl of protein C activator (Protac, 2 μM FP31_W215A or 2 μM FP31_WE) for 5 min at 37 °C in a 96-well plate. The amount of activated protein C was quantified by adding 125 μL of a solution of chromogenic substrate S-2366 (2.4 mM) (Diapharma Group, OH) and thrombin specific inhibitor argatroban (160 μM) (Sigma-Aldrich, MO) for 5 min at 37 °C. The reaction was quenched by addition of 150 μl citric acid and read at 405 nm. Mixing human control normal plasma with sterile saline solution to give 0, 25, 50 and 100% protein C generated standard calibration curves reported in Fig. 5A. Healthy control plasma was purchased from Innovative Research, MI and human abnormal control plasma depleted of protein C (ref A223301) was purchased from Biophen, Aniara Diagnostica, OH.