Figure 2.

ADP priming in the presence of LPS upregulates surface HMGB1 expression. Washed platelets from 8 dogs were unstimulated (resting) or activated with 5 μg/ml LPS or 10 μM ADP. Platelets were also primed with 10 μM ADP prior to treatment with LPS. (A,B) Surface HMGB1 expression from 8 dogs was assessed by flow cytometry and measured as percent (%) positive or median fluorescence intensity (MFI) fold change on a log₁₀ scale. While LPS alone did not augment surface HMGB1 expression, LPS in the presence of ADP upregulated HMGB1 expression. (C,D) Total cellular HMGB1 expression in 1 ×10⁸ platelets/ml was further assessed by western blot and relative densitometry calculated based on beta-actin, used as loading control in 6 dogs with 2 replicates in each dog with identical results. (C) Representative immunoblot of HMGB1 (~25–29 kDa) and β-actin (~42 kDa) in platelet lysate after stimulation with 10 μM ADP, 5 μg/ml LPS or LPS in the presence of ADP. Thrombin (0.01 U/ml) served as positive control. LPS alone or LPS in the presence of ADP did not upregulate total cellular HMGB1 expression. Each box represented the 25th and 75th quartiles and line represents the median. Whiskers represent the range of data. *p > 0.05. One-way repeated measures ANOVA was used to assess the effect of platelet activation on HMGB1 expression, followed by post-hoc analysis by Tukey multiple comparisons.