PGN from gram-positive bacteria activates human platelets. (A) Aggregation traces for (line A) platelet-rich plasma + thrombin (1 U/mL); (line B) PGN preincubated in plasma for 30 minutes at 37°C and then added to platelet-rich plasma; (line C) platelet-rich plasma + PGN (10 μg/mL). For line C, the final concentration of PGN was 10 μg/mL and of plasma was 10% v/v. (B) PGN (0-40 μg/mL) preincubated with plasma as described in panel A or thrombin (1 U/μλ) was added to platelets, and percentage of light transmission was monitored for 15 minutes. Data are reported as means of the percentage of light transmission of 4 independent experiments from different donors. (C-J) Washed platelets were stimulated with (C,G) Tyrode buffer, (D) thrombin, (H) ionomycin, (E,I) 10 μg/mL PGN in Tyrode buffer, and (F,J) 10 μg/mL PGN preincubated with plasma at 37°C for 15 minutes. After fixation, platelets were identified by CD41 and analyzed (C-F) for αIIbβ3 upregulation by PAC-1 or (G-J) PS exposure by annexin V. The data in C through J are representative of >10 experiments. (K) Platelet-rich plasma was pretreated with tirofiban (1 μM) to prevent platelet aggregation. PGN or ionomycin (10 μM) were added to platelet-rich plasma for 30 minutes at 37°C. After fixing and staining, annexin V–positive platelets were examined by flow cytometry. (L) The exposure of anionic phospholipids on the platelet surface was analyzed by a continuous prothrombinase activity assay. Platelets were incubated with buffer (open circles), 10 μg/mL PGN (squares), plasma-preincubated PGN (diamonds), or 10 μM ionomycin (closed circles). Thrombin generation was followed over time using the chromogenic substrate Spectrozyme TH. A representative experiment performed in triplicate is shown after normalization to the maximum conversion of the substrate.