P2Y purinoceptor stimulation increases ARF6-GTP levels in human platelets.

<p>(<b>A</b> and <b>B</b>) Human platelets were treated with ADP (10 µM; 0–5 min) and endogenous ARF-GTP levels assessed by using GST-GGA3 VHS-GAT pull down assay. Total ARF6 (<b>A</b>) or ARF1 (B) expression in platelet cell lysates and ARF6-GTP (A) or ARF1-GTP (B) precipitated with GST-GGA3 VHS-GAT beads were detected by immunoblotting using an anti-ARF6 mouse monoclonal and an anti-ARF1 rabbit polyclonal antibodies. The relative intensity of each ARF6-GTP or ARF1-GTP band was normalised to total ARF6 or ARF1 measured by densitometry (<b>A</b> and <b>B</b>). Values are mean ± SEM from three separate experiments. *p<0.05 compared with no ADP treatment alone control (Mann–Whitney <i>U</i>-test). (<b>C</b>) Human platelets were treated with thrombin (0.3 unit/ml) and collagen-related peptide (CRP; 5 µg/ml) for 0–5 mins and and endogenous ARF-GTP levels assessed. The data are representative of three independent experiments (<b>D</b>) Platelets were incubated with SecinH3 (15 µM) or vehicle alone for 30 minutes. Platelets were then treated with ADP (10 µM; 0–5 min) and endogenous ARF6-GTP levels assessed. The data represent mean ± SEM of three independent experiments. *p<0.05 compared with no ADP treatment alone control (Mann–Whitney <i>U</i>-test).