APC-catalyzed inactivation of platelet-derived factor Va bound to thrombin-activated platelets or centrifugation-induced platelet microparticles. Platelets (1 × 109/mL) in the presence of RGDS peptide (1 mmol/L) from a normal individual were treated with 2 NIH U/mL (20 nmol/L) of α-thrombin for 5 minutes to both activate the platelet and release and activate platelet-derived factor Va. Hirudin (30 nmol/L) was then added to inhibit thrombin. The thrombin-activated platelets were either used as the required membrane surface (•; initial cofactor concentration, 2.5 nmol/L) or platelets were centrifuged out of solution (1,100g for 5 minutes) generating platelet microparticles that presumably provided an adequate membrane surface (▪; initial cofactor concentration, 1.9 nmol/L). APC (0.25 nmol/L) was then added and at selected time points residual cofactor activity was monitored as described in Fig 1. The data points were normalized to the initial concentration of released cofactor for each donor. Upon stabilization of the platelet-derived cofactor activity on thrombin-activated platelets (∼120 minutes), 20 μmol/L PCPS vesicles were added (arrowhead) to the platelet membrane/platelet-derived factor Va/APC mixture and a substantial loss of platelet-derived cofactor activity was observed after 30 minutes of incubation (▴).
