SPR-observed adsorption of primary and secondary fibrinogen layers. A primary layer was formed by the adsorption of fibrinogen onto the gold surface, followed by washing with TBS and PBS; a secondary fibrinogen layer was formed by the immobilization of fibrinogen onto the primary layer, which was treated with thrombin to convert the adsorbed fibrinogen into fibrin. Arrows indicate the replacement of solutions. Curve 1, fibrinogen (Fbg) 500 μg/mL, TBS, PBS; thrombin 2.5 U/mL (THR), PBS, TBS; mixture of PPACK 10μM and hirudin 6 U/mL, TBS; fibrinogen 200 μg/mL, TBS. Curve 2, fibrinogen 20 μg/mL, TBS, PBS; thrombin 2.5 U/mL, PBS, TBS; PPACK 10μM, hirudin 6 U/mL, TBS; fibrinogen 200 μg/mL, TBS. Curve 3, fibrinogen 20 μg/mL, TBS, PBS, TBS; fibrinogen 200 μg/mL, TBS. Curve 4, fibrinogen 20 μg/mL, TBS, PBS; thrombin 2.5 U/mL, PBS, TBS; PPACK 10 μM and hirudin 6 U/mL, TBS; fibrinogen 20 μg/mL, TBS.