Sirt3 deficiency had a minor impact on platelet functions.

<p>Platelet aggregation was induced by (A) collagen, (B) thrombin and (C) ADP at the concentration described using (A), (B) washed platelets or (C) platelet-rich plasma (PRP) from Sirt3-/- mice and WT mice. Representative aggregation traces are on the right. n = 7–10. (D) Washed platelets from Sirt3-/- mice and WT mice were incubated with thrombin 25 or 100 mU/mL for 20 min at 37°C. Percentage of JON/A and P-selectin double positive cells were quantified by flow cytometry. n = 4.*<i>P</i><0.05 vs WT (Student’s t tests).</p

No difference was observed in <i>in vitro</i> NETosis between Sirt3-/- and WT.

<p>Peripheral neutrophils isolated from Sirt3-/- mice and WT mice were stimulated with (A) PMA (25, 100 nM, 4h) or (B) ionomycin (4 μM, 2h). The percentage of neutrophil extracellular traps (NET) generation was evaluated. n = 7, *<i>P</i><0.05 vs WT (Student’s t tests). Fluorescent images of NET formation after PMA (25, 100 nM, 4h) or ionomycin (4 μM, 2h) stimulation and Hoechst staining of peripheral neutrophils isolated from (C) WT mice and (D) Sirt3-/- mice (Scale bar: 20 μm). Arrows indicate NETs.</p

Deficiency of Sirt3 in platelets increased mitochondrial ROS production.

<p>(A) Western blot analysis using washed platelets from Sirt3-/- mice or WT mice. Photograph of a representative blot of experiments is shown. (B), (C) Flow cytometric determination of mitochondrial and cytosolic ROS in platelets. Washed platelets were incubated with thrombin (100 mU/mL) or vehicle for 30 min at 37°C in the presence of (B) MitoSox or (C) dihydrorhodamine-123. MitoSox or Rhodamine positive platelets were quantified by flow cytometry for each condition. n = 6, *<i>P</i><0.05 vs WT (Student’s t tests).</p

Deficiency of Sirt3 did not affect venous thrombosis <i>in vivo</i>.

<p>Sirt3-/- mice and WT mice underwent inferior vena cava (IVC) stenosis for 3 hrs. (A) Incidence of thrombus formation, (B) weight and (C) length are presented. (D) Thrombin anti-thrombin (TAT) complex, (E) plasma DNA concentration or (F) plasma soluble P-selectin concentration were determined. Circulating (G) platelet and (H) neutrophil counts were evaluated before (0 hr) and after (3 hrs) IVC stenosis. n = 11–12, *<i>P</i><0.05, **<i>P</i><0.01 vs WT or corresponding group (Student’s t tests). Thrombus frequencies were analyzed using χ2 tests of contingency tables.</p

Deficiency of Sirt3 augmented production of ROS in neutrophils.

<p>(A) Western blot analysis using bone marrow neutrophil lysates from Sirt3-/- mice or WT mice. Photograph of a representative blot is shown. (B) and (C) represent flow cytometric determination of mitochondrial and cytosolic ROS in neutrophils. Diluted anti-coagulated whole blood from Sirt3-/- mice and WT mice was incubated with (B), (C) PMA (25, 100 nM), (C) ionomycin (4 μM) or vehicle for 30 min at 37°C in the presence of (B) MitoSox or (C) dihydrorhodamine-123. MitoSox or Rhodamine-positive neutrophils were quantified by flow cytometry for each condition. n = 5–7. *<i>P</i><0.05 vs WT (Student’s t tests).</p