APC-catalyzed inactivation of platelet-derived factor Va and factor Va^Leiden bound to phospholipid vesicles. Platelets (1 × 10⁹/mL) were treated with 5 NIH U/mL (50 nmol/L) of -thrombin for 5 minutes to both activate the platelets and release and activate the platelet-derived factor V. Hirudin (60 nmol/L) was then added to inhibit thrombin. The activated platelets were immediately removed from suspension by gentle centrifugation (1,100g, 5 minutes), and PCPS vesicles (20 μmol/L) were added to the supernatant to provide an appropriate alternate anticoagulant surface. APC (0.25 nmol/L) was then added. At selected time points, samples of the reaction mixture were withdrawn and subjected to SDS-PAGE using a 5% to 15% gradient gel. After transfer to nitrocellulose, fragments were visualized using a monoclonal antibody, HFVa_HC#17, as described.25 Each of the panels represents the inactivation of secretable platelet-derived factor Va on phospholipid vesicles by APC: (A) normal platelet-derived factor Va, (B) platelet-derived factor Va derived from a heterozygous factor V^Leiden individual, (C) platelet-derived factor Va derived from JMW, and (D) platelet-derived factor Va derived from FW. The time course of inactivation by APC is given at the top of each immunoblot. In (A) and (C), 145* indicates the platelet factor Va/APC mixture after a 145-minute incubation, with an additional 20 nmol/L APC incubated for 5 minutes. In (B) and (D), following a 45-minute incubation with 0.25 nmol/L APC, platelet-derived factor Va was incubated with increasing concentrations of APC (2, 10, 20, 40 nmol/L) for an additional 15 minutes, which is indicated in the last four lanes of these immunoblots. The position of the molecular weight markers are indicated at the left of the immunoblots. Arrows to the right of the immunoblots represent residue numbers corresponding to factor Va fragments derived from APC-induced cleavage.