Human platelets treated with the peptide RGDW show a fibrin-mediated delayed wave (DW) of increased light transmission when activated with Thr; fibrinogen enhances the DW, and platelet activation is required for interaction with polymerized fibrin. Washed platelets (2 × 10⁸/mL) were treated with 150 μM RGDW and/or the fibrin polymerization inhibitor Gly-Pro-Arg-Pro (GPRP) for 20 minutes at room temperature and then activated with either (A) 25 μM of a Thr PAR-1 receptor activating peptide (SFLLRN; T6) or (B) 0.2 U/mL Thr in an aggregometer cuvette. Changes in light transmission were measured at 37°C with stirring. (C) Quantitation and statistical analysis of the corresponding maximal change in light transmission (ΔLT) of the T6- or Thr-induced initial wave (<2 minutes after agonist injection) in the presence or absence of RGDW (left panel), and Thr-induced DW (>2 minutes after agonist injection), presented as mean ± SD. Where indicated, samples were treated with human fibrinogen (10, 50, and 100 µg/mL; red dots), GPRP (5 µM; green dots), or mAbs 6D1 or 6F1 (both at 10 µg/mL; blue dots). Data are presented as mean ± SD. Statistical analyses were performed using Student t test between samples tested in the same experiments with and without the indicated intervention (color-coded). *P < .05; ****P < .0001. ns, nonsignificant. (D) In parallel studies, the reaction was stopped at the indicated times by adding one-third volume of 3× sodium dodecyl sulfate (SDS) sample buffer. Samples were subjected to SDS-polyacrylamide gel electrophoresis under reducing conditions and immunoblotted with an mAb specific for the fibrinogen γ-chain. (E) Addition of fibrinogen at 10 µg/mL (blue tracing), 50 µg/mL (purple tracing), 100 µg/mL (black tracing) enhanced the DW. (F) Effect of added fibrinogen on the time to DW onset. Fibrinogen at the indicated concentrations was added to washed platelet samples treated with 150 μM RGDW in an aggregometer cuvette at 37°C with stirring. After 60 seconds, 0.2 U/mL Thr was added (time of injection), and the time of DW onset was recorded. Data were presented as mean ± SD. Significant differences (P < .05) between groups were estimated using Student t test. *P < .05, **P < .01. (G) Washed platelets were treated with 15 μg/mL PT25-2 for 20 minutes at room temperature. Then, soluble fibrin (100 μg/mL) was added, and the change in light transmission was monitored. All tracings and the immunoblot are representative of at least 3 independent experiments.