Factor Xa– and LMWH-based assays for the APC-independent activity of protein S in plasma. Normal pooled plasma was incubated for 30 minutes with (dashed line) or without (solid line) anti–protein S neutralizing antibodies. (A) Coagulation was triggered with CaCl₂ (5 mM), factor Xa (20 pM), and phospholipids (6 μM DOPS/DOPC, 20/80 M/M) at indicated final concentrations. (B) LMWH was added and coagulation was triggered with dilute human recombinant tissue factor as described in “Patients, materials and methods.” Fibrin formation was followed by measuring the change in turbidity at 405 nm. (C) Protein S–dependent anticoagulant ratio (PSdAR) of the times to half-maximal turbidity at 405 nm in the absence and presence of anti–protein S antibodies. The factor Xa–based assay (•) and LMWH-based assay (▾) are represented as a function of assay duration.