Fig 1: GPVI and TLR2 share common signaling nodes and cooperate to potentiate platelet activation. (A) Replicate preparations of washed human platelets (1x109 ml) were activated with Pam2CSK4 (10 µg/ml) or CRP-XL (2 µg/ml; 5 min) and cell lysates analyzed by immunoblot for the indicated antibodies; n=3 independent experiments. (B, D) Platelets (2×108/ml) were incubated with a luciferin luciferase reagent to measure ATP release and dense granule secretion. Data are expressed as raw mean luminescence (a.u.); n=4 experiments. (C, E) Platelets (5×107/ml) were pre-treated and activated as above in the presence of fluorophore-conjugated antibodies against P-selectin (CD62P-APC) or the activated conformation of human integrin aIIbß3 (PAC1-FITC) and subsequent analysis by flow cytometry; n=3-6 experiments. Data are presented as total mean fluorescence intensity (MFI). (F) Platelet lysates were prepared as in A and analyzed by Western Blot for DAPP1 activation; representative of n=3 experiments. (G) Cover glass was coated with soluble collagen (50 µg/ml), blocked with BSA, and incubated with replicate samples of washed human platelets (2x107/ml) untreated or stimulated with Pam2CSK4. After 45 min (37°C), cover glass was washed with PBS and adherent platelets were imaged with DIC microscopy; n=5 experiments. Pam2CSK4 was used at 10 µg/ml (A–C, F, G) or 5 µg/ml (D, E). CRP-XL was used at 2 µg/ml. Scale bar indicates 10 µm. * indicates statistical significance (p<0.05) compared to Pam2CSK4 (B, C) or to untreated (D–G). Error bars indicate standard error of the mean (SEM).