Kinetics of thrombin-mediated release of fibrinopeptides from the secondary layer of surface-bound fibrinogen-fibrin complexes. All fibrinogen-fibrin complexes were formed by the immobilization of the secondary fibrinogen layer on thrombin-treated (2.5 U/mL of thrombin for 60 minutes, followed by a mixture of PPACK and hirudin for 20 minutes) primary fibrinogen layers adsorbed onto polystyrene. (A) The primary layer was adsorbed from 500 μg/mL of fibrinogen for 30 minutes; the secondary layer was immobilized at 200 μg/mL of fibrinogen for 60 minutes. (B) The primary layer was adsorbed from 20 μg/mL of fibrinogen for 150 minutes; the secondary layer was immobilized at 20 μg/mL of fibrinogen for 120 minutes. (C) The primary layer was adsorbed from 20 μg/mL of fibrinogen for 150 minutes; the secondary layer was immobilized at 200 μg/mL of fibrinogen for 60 minutes. The amounts of FpA (□) and FpB (■) released from the secondary layers upon incubation with 2.5 U/mL of thrombin were determined by HPLC, as described in “Methods.” Each point represents a mean value obtained from 3 independent experiments.