Fig 2: Differential roles of SFKs (Src family kinases) and Syk in agonist-induced p47phox phosphorylation. A and B, Washed mouse platelets were treated with the SFK inhibitor PP2 (10 µmol/L) or negative control compound PP3(10 µmol/L), and stimulated with (A) 0.025 U/mL thrombin or (B) 1 µg/mL CRP (collagen-related peptide) to induce aggregation. C and D, Washed mouse platelets were treated with DMSO control (ctrl) or the Syk inhibitor piceatannol (15 µmol/L) and stimulated with (C) 0.025 U/mL thrombin or (D) 1 µg/mL CRP to induce aggregation. After 2 min, platelets were lysed and analyzed via SDS-PAGE and Western Blot for phosphorylated p47phox at Ser304 (p-p47phox S304) or Ser328 (p-p47phox S328), total p47phox or α-tubulin as loading control. The blots were scanned and quantified using National Institutes of Health Image J and plotted as mean±SEM (*P<0.05, **P<0.01, and ***P<0.001, Student t test comparing inhibitor-treated samples with controls, n=3). FL indicates fluorescence.

Fig 3: Reactive oxygen species (ROS) production and p47phox activation during platelet spreading on integrin ligand fibrinogen. A, Images of DCF fluorescence in mouse platelets loaded with 10 µmol/L carboxy-H2DCFDA and allowed to spread on fibrinogen, with or without 0.01 U/mL thrombin. N-acetylcysteine (NAC)-treated platelets was used as negative control (scale bar, 10 µm). B, Quantification of the total ROS production per cell was calculated by integrating the fluorescence (FL) intensity (mean±SEM, *P<0.05, ***P<0.001, 1-way ANOVA with Dunn multiple comparisons (Fg, -NAC: n=75, Fg, +NAC: n=19; Fg+thrombin, -NAC n=95, Fg + thrombin, +NAC n=47). C, Washed mouse platelets spreading on fibrinogen in the absence or presence of MnCl2 (0.5 mmol/L) or (collagen-related peptide [CRP]; 1 µg/mL) were lysed at the indicated times and analyzed via SDS-PAGE and Western Blot for phosphorylated p47phox at Ser304 (pS304-p47phox), total p47phox, or a-tubulin as loading control. D, Quantification of the Western blot results for phosphorylated p47phox at Ser304 (pS304-p47phox; mean±SEM, n=4). E, Washed wild type (WT) and ß3-/- mouse platelets, either resting or spreading on fibrinogen in the absence or presence of MnCl2 (0.5 mmol/L) or CRP (1 µg/mL), were lysed after 30 min and Western Blot for phosphorylated p47phox at Ser304 (pS304-p47phox), total p47phox, or a-tubulin as loading control. F, Quantification of (E), plotted as mean±SEM (*P<0.05, **P<0.01, ***P<0.001, 1-way ANOVA with Tukey multiple comparison test, n=4).

Fig 4: Ga13-mediated integrin outside-in signaling stimulates reactive oxygen species (ROS) production, p47phox activation and Syk phosphorylation. A, Images of DCF fluorescence in mouse platelets loaded with 10 µmol/L carboxy-H2DCFDA, treated with 100 µmol/L scrambled control peptide or mP6, and allowed to spread on fibrinogen with or without 0.01 U/mL thrombin (scale bar, 10 µm). B, Quantification of the total ROS production per cell in (A) was calculated by integrating the fluorescence (FL) intensity (mean±SEM, Kruskal-Wallis test with Dunn multiple comparisons, Fg -thrombin, Scr: n=100, Fg -thrombin, mP6: n=6; Fg+thrombin, Scr: n=45, Fg+thrombin, mP6 n=9). C and D, Washed mouse platelets were treated with 100 µmol/L scrambled control peptide or mP6, allowed to adhere and spread on fibrinogen, and lysed for (C) SDS-PAGE and Western blot for phosphorylated p47phox at Ser304 (pS304-p47phox), total p47phox or a-tubulin as loading control. D, Quantification of (C), plotted as mean±SEM, Student t test, n=4). E and F, Washed mouse platelets, treated with 100 µmol/L scrambled control peptide or mP6 (E), or wild type and Ga13-/- platelets (F), were preincubated with 10 µmol/L carboxy-H2DCFDA and stimulated with 0.025 U/mL thrombin under the shear rate of 800/s, and total ROS production is shown as mean±SEM (Student t test, n=3). G and H, Mouse platelets treated with scrambled control peptide or mP6 were stimulated with thrombin (0.025U/mL) for increasing lengths of time and then immunoblotted for Syk phosphorylated at Tyr519/520, total Syk and tubulin. G, A typical blot; (H) quantification (n=3). I and J, Mouse platelets treated with ROS inhibitors apocynin (500 µmol/L) and NAC (1 mmol/L) were stimulated with thrombin (0.025 U/mL) for increasing lengths of time and then immunoblotted for Syk phosphorylated at Tyr519/520, total Syk, and tubulin. I, A typical blot; (J) quantification (mean±SEM, t test, n=3). *P<0.05, **P<0.01, and ***P<0.001.