Bacteria-induced platelet aggregation is modulated by PF4 and heparin and is impaired in GPS platelets. (A) PF4 binding to bacteria. Bacteria were incubated with 20 μg/mL biotinylated human PF4 in buffer (i) or in the presence of 46% plasma from healthy donors (ii). After a secondary incubation with peridinin chlorophyll protein-Cy5.5–conjugated streptavidin, PF4 binding was analyzed by flow cytometry (Cytomics FC 500; Beckman Coulter). The GMFI multiplied by the percentage of labeled bacteria constituted binding activity. Results shown are mean ± SD of 3 independent experiments; *P < .05. (B) Effect of PF4 on the lag time for onset of bacteria-induced platelet aggregation. Washed platelets were supplemented with fibrinogen (1 mg/mL) and hIgGs (0.1 mg/mL). Purified human PF4 (20 μg/mL) and bacteria were added simultaneously to the platelet preparation and aggregation was recorded (S sanguinis 133-79, n = 2; S aureus Newman, n = 2; S gordonii DL1, n = 6; S oralis CR834, n = 3; S pneumoniae IO1196, n = 2). (C) Effect of heparin on bacteria-induced platelet aggregation. Aggregation reactions were performed in plasma in the presence of varying doses of UFH: 0.06 μg/mL (0.0125 U/mL), 0.6 μg/mL (0.125 U/mL), 6 μg/mL (1.25 U/mL), 60 μg/mL (12.5 U/mL), 600 μg/mL (125 U/mL), 3000 μg/mL (625 U/mL). UFH was preincubated with platelets for 2 minutes before adding bacteria. Changes in lag time in UFH-treated reactions are presented as the mean ± SEM; *P < .05 (S sanguinis 133-79, n = 6; S aureus Newman, n = 5; S gordonii DL1, n = 6; S oralis CR834, n = 5; S pneumoniae IO1196, n = 6). For S sanguinis 133-79, S gordonii DL1, and S pneumoniae IO1196, 3000 μg/mL UHF inhibited aggregation in all donors (not shown). Lag times (mean ± SD) in the untreated reactions were: S sanguinis 133-79, 273 ± 180 seconds; S aureus Newman, 218 ± 101 seconds; S gordonii DL1, 528 ± 136 seconds; S oralis CR834, 206 ± 82 seconds; S pneumoniae IO1196, 410 ± 152 seconds (see also supplemental Figure 4). (D) Bacteria-induced platelet aggregation in GPS and control platelets. The final platelet concentration in GPS PRP was 1 × 10⁸ platelets per mL and, therefore, the control PRP was diluted in buffer to obtain the same concentration. ADP (30μM) was used as a control agonist for platelet aggregation. Two duplicates were performed per each reaction and sample, and 1 representative trace is shown. Lag times for the reactions shown are: S sanguinis 133-79, 2 minutes; S aureus Newman, 7 minutes 10 seconds; S gordonii DL1, 20 minutes; S oralis CR834, 6 minutes (GPS platelets, 4 minutes); S pneumoniae IO1196, 8 minutes. GMFI, geometric mean fluorescence intensity.