Screening of protein S variants for APC cofactor activity. The APC cofactor activity of protein S was evaluated at 16nM APC and 100nM protein S by CAT. The peak height in the absence of protein S was set to 100%. A high concentration of APC, leading to almost complete inhibition of thrombin generation with 100nM WT protein S, was chosen specifically for screening purposes as this allows widening of the assay window at which mutants with reduced APC cofactor activity are visualized. Results were confirmed by evaluating protein S (at 60 and 90nM) cofactor activity toward 4 or 9nM APC.