APC-catalyzed inactivation of platelet-derived factor Va and factor Va^Leiden bound to phospholipid vesicles. Platelets (1 × 10⁹/mL) from normal (n = 3) or homozygous factor V^Leiden individuals (n = 3) were treated with 5 NIH U/mL (50 nmol/L) of α-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 for 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 added and at selected time points residual cofactor activity was monitored in a prothrombinase assay using purified protein components with saturating amounts of factor Xa (5 nmol/L) and PCPS vesicles (20 μmol/L) as previously described.8,36 At the same time intervals, samples of the reaction mixture were withdrawn and subjected to SDS-PAGE using a 5% to 15% gradient gel.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 an MoAb (αHFVa_HC#6), as described,25,29,44 that recognizes an epitope on the heavy chain of factor Va between amino acids 307-506. The line drawn through the inactivation profiles (A and B) represents the average of the three donors at each given time point and does not represent an attempt to fit the data to a first-order rate equation. The data points were normalized to the initial concentration of released cofactor for each donor. In (A), each of the symbols represents the time-dependent, APC-catalyzed inactivation of the platelet-derived factor Va cofactor activity from three normal donors (initial cofactor concentrations: 0.88, 0.91, and 2.70 nmol/L). The inset represents the proteolytic fragments derived from the inactivation on PCPS vesicles as visualized using immunoblotting techniques. Lane 1, platelet-derived factor Va, no APC; lanes 2 through 9, membrane-bound platelet-derived factor Va with APC for 1, 5, 10, 15, 30, 60, 90, and 120 minutes. In (B), each of the symbols represents the time-dependent, APC-catalyzed inactivation of the platelet-derived factor Va^Leiden cofactor activity from three factor V^Leiden donors (initial cofactor concentrations: 0.81, 0.91, and 1.30 nmol/L). The inset represents the proteolytic fragments derived from the inactivation on PCPS vesicles. Lane 1, platelet-derived factor Va^Leiden, no APC; lanes 2 through 10, membrane-bound platelet-derived factor Va^Leiden with APC for 1, 5, 10, 15, 30, 60, 90, and 120 minutes. The position of the molecular weight markers are indicated at the left of the insets. Controls here, and in other experiments, indicated that, in the absence of APC, platelet-derived factor Va and factor Va^Leidenretained full cofactor activity throughout the time course either in the presence of PCPS vesicles or thrombin-activated platelets.