APC-catalyzed inactivation of plasma-derived factor Va subsequent to clot formation in the presence of PCPS vesicles or platelets. Pooled normal human plasma was diluted (1:10) in a glass test tube with 20 mmol/L HEPES/0.15 mol/L NaCl, pH 7.4. Phospholipid vesicles (PCPS; 10 μmol/L; A and C) or washed normal human platelets (1 × 10⁸/mL; B and D) were added. CaCl₂ (5 mmol/L, final) was then added to initiate clot formation, which was observed visually. In (C) and (D), exogenous APC (2.0 nmol/L) was added subsequent to clot formation as indicated by the arrow above the blots. At selected time intervals (indicated above each gel), samples of the reaction mixture were analyzed by SDS-PAGE and Western blotting techniques with MoAb α-HFVa_HC#17, as described.45 The position of the molecular weight markers are indicated at the left of each blot and residue numbers corresponding to factor Va fragments are given at the right of each blot. Fragments migrating at approximately 45 kD and approximately 40 kD, which represent amino acids 307-709 and 307-679, respectively, normally migrate at approximately 60 kD and approximately 54 kD. However, because we are working with dilute plasma, the mobility of these fragments appears increased because of the high concentration of albumin present.