Both PGN and plasma factors contribute to platelet activation. PGN was preincubated in plasma for 1 hour and then centrifuged at 2000g to separate the particulate PGN pellet and PGN-activated plasma supernatant. The washed platelets were stimulated independently with PGN preincubated with plasma (PGN + Plasma, B and F), the PGN pellet (PGN Pellet, C and G) or PGN-activated plasma supernatant (Supernatant, D and H) before staining for (A-D) PAC-1 or (E-H) annexin V and analysis by flow cytometry. (I) Quantitative and statistical data are shown for 3 replicates of experiments performed as in panels A through H. *P < .05 and NS, not significant for comparisons of no stimulation (NS) with PGN + plasma, PGN pellet, or supernatant.