Appendix Table of Contents. 1. Appendix Figure legends S1-S13 and Appendix Table S1 and S2. 2. Appendix Figures S1-S13

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1 Appendix Table of Contents. Appendix Figure legends S-S3 and Appendix Table S and S. Appendix Figures S-S3

2 . Appendix Figure legends S-S3 and Appendix Table S and S Appendix Figure S. Western blot analysis of oxidative adducts Oxidative adducts (3-nitrotyrosine (3-NT), aldehyde adducts (4-HNE), carbonyl derivatives (dinitrophenyl (DNP)-derivatized carbonyl)) and polyubiquitination in pooled (n=4) and pooled (n=8) platelet protein lysates. GAPDH was used as the loading control. Quantification analysis of each oxidative adducts were performed using the Image J program. Appendix Figure S. High magnification EM of platelet from and samples. EM of platelets from and patients demonstrating increased vacuolation in platelets. Appendix Figure S3. Mitophagy is induced in platelets. A. Triple staining for CoxIV, LC3, and LAMP (lysosome) in and platelets. A representative enlarged single platelet is shown. Arrow indicates positive signal for LC3 and LAMP. Graph indicates colocalization between LC3, LAMP and CoxIV signal. The y-axis indicates fold change of colocalization between LC3 and LAMP in the mitochondria signaling area (p<.5 vs. ). Signal intensity of each group was converted to fold compare with values. B. Representative Western blot analysis showing increased Parkin and LC3 protein expression in platelet mitochondria fraction. C. Immuno-EM of samples () demonstrating multiple clusters and colocalization between LC3 and Parkin. Red arrow indicates immunogold-labeled LC3 antibody ( nm) and black arrow indicates immunogold-labeled Parkin antibody (5nm). Black bars in the inset represent nm. D. Triple staining for CoxIV, LC3, and Parkin (mitophagy) in and platelets. A representative enlarged single platelet is shown. The arrows indicate positive signal for LC3 and

3 Parkin. Graph indicates colocalization between LC3, Parkin, and CoxIV signal. The y-axis indicates fold change of colocalization between LC3 and Parkin in mitochondria (CoxIV) (p<. vs. ). Signal intensity of each group was converted to fold and compared with values. Appendix Figure S4. Western blot analysis of Bnip3/Nix in and platelets. A. Representative Western blot analysis of Bnip3/Nix, Parkin and LC3II in (#7-9) and (#9-6) platelets. B. Graph indicate quantification Bnip3/Nix, Parkin and LC3II in (#7-9) and (#9-3) platelets. (p<., p<.5 vs. ). GAPDH served as a loading control. Appendix Figure S5. qpcr analysis of mitochondrial content in human and mice platelets. A. Representative qpcr analysis of NADH dehydrogenase subunit (ND) and 6sRNA in human platelets (=3 and =3). 8sRNA was used for normalization. (p<., p<.5 vs. ). qpcr primers used for human : ND F: 5 -ACG CCA TAAAAC TCT TCA CCA AAG- 3,ND R: 5 -TAGTAG AAG AGC GAT GGT GAG AGC TA-3,6s RNA F: 5 - GAACCAGACGAGCTACCTAAG-3 and 6s RNA R: 5 - GGTTTGTCGCCTCTACCTATAAA-3 B. Representative qpcr analysis of NADH dehydrogenase subunit (ND) in mouse platelets (=3 and =3). 8sRNA was used for normalization. (p<., p<.5 vs. ). qpcr primers used for mouse platelets: ND F: 5 - TGCACCTACCCTATCACTCA-3 and ND R: 5 -GGCTCATCCTGATCATAGAATGG -3 3

4 Appendix Figure S6. Western blot analysis of Ulk activation and mtor downstream targets in and platelets. A. Representative Western blot analysis of phosphorylation of Ulk (S757) and total Ulk in (#4-6) and (#9-8) platelets. Graph indicates quantification of phosphorylation of Ulk (S757) and total Ulk (p<., p<.5 vs. ). GAPDH served as a loading control. B. Representative Western blot analysis of phosphorylation of p7s6k and S6 in (#7-9) and (#9-6) platelets. Graph indicates quantification of phosphorylation of p7s6k and S6 in (#7-9) and (#9-3) platelets. (p<., p<.5 vs. ). GAPDH served as a loading control. Appendix Figure S7. p53 activity does not affect ROS induced autophagy. A. platelets were treated with H O to induce autophagy and treated with or without PFT-α (p53 inhibitor, and μm) to assess for inhibition of autophagy. This Western blot analysis is representative of three independent experiments. B. Quantification of the three independent experiments is provided at right. (p<., p<.5 vs. or H O group, n=3 for each group. C. Triple staining with Mitotracker, CoxIV, and LC3 in and H O with or without μm PFT-α treated platelets using confocal microscopy. Appendix Figure S8. Mitophagy induced in human platelet and maybe protective A. Triple staining for CoxIV, LC3, and Parkin in platelets. Treatments were performed with high glucose (HG) and/or CCCP. Enlarged areas are provided showing areas of colocalization (CCCP treatment, three arrows in proximity). All images are representative of 5 images taken randomly and repeated three times in independent experiments. 4

5 B. Graph indicates colocalization between LC3, Parkin and CoxIV signal. The y-axis indicates fold change of colocalization between LC3 and Parkin in the mitochondria (CoxIV). Signal intensity of each group was converted to fold and compared with values (p<. vs. or HG group). C. ΔΨm and platelet apoptosis were measured by flow cytometry analysis in, HG, HG with H O, or HG with H O and 3MA using human platelets. ΔΨm was detected using TMRM and apoptosis level was assessed with Annexin-V (PS externalization). Graph indicates the percentage of TMRM negative cell in the total cell population (p<.vs. HG or HG with H O, NS means no significance). D. Graph indicates the percentage of Annexin V positive cell in total cell population (p<. vs. HG with H O, NS means no significance). Appendix Figure S9. Human platelet mitophagy protects in. The recognized autophagy inhibitor (μm Spautin- for h) was used to treat platelets and compared to assessing for phosphorylated p53, p53, and LC3I/II. Graph indicate quantification of phosphorylation of p53 (S5) and LC3II (p<.5 vs. ). GAPDH served as the loading control. Appendix Figure S. Mitophagy reduces platelet aggregation. Platelet suspensions were incubated with CCCP for hr. Platelet aggregation was monitored at 37 C with constant stirring ( rpm) in a dual-channel lumi-aggregometer (model 7; Chrono-Log). Platelet aggregation was measured as the increase in light transmission for minutes, starting with the addition of μl of mg/ml collagen (Chrono-Log) into 5 μl as a proaggregatory stimulus; the final concentration was 4 μg/ml. The maximum aggregation was expressed as a percentage of maximum light transmission, with nonstimulated PRP being % and PPP %. 5

6 A. platelet suspensions were incubated with or without CCCP for hr. Percentage of light transmission was measured in platelet suspensions under SO (black line) and CCCP (Blue line) in response to 4 μg/ml collagen for minutes (n = 4 in each group). B. Western blot analysis and quantitation analysis using 4 μg/ml collagen treatment samples with or without CCCP. C. Graph indicates quantification of LC3II. (p<.5). GAPDH served as a loading control. Appendix Figure S. Platelet apoptosis in PINK knockout mouse platelets. A. Western analysis in WT and PINK -/- demonstrating the inability to induce LC3 above basal levels despite the addition of H O. Shown graphically are the LC3II expression level (p<.5, n=3) B. ΔΨm and platelet apoptosis were measured by flow cytometry analysis in WT, PINK-/-, WT treated with H O or PINK-/- treated with H O. ΔΨm was detected using TMRM (p<., n=3 for each group) C. Apoptosis level was assessed with Annexin-V (PS externalization). Graph indicates the percentage of Annexin V positive cell in the total cell population (p<., p<.5, NS no significance, n=3 for each group) D. ΔΨm and platelet apoptosis were measured by flow cytometry analysis in WT, Parkin -/-, PINK-/-, WT treated with H O or Parkin-/- treated with H O or PINK-/- treated with H O (.5 or mm). ΔΨm was detected using TMRM (p<., n=3 for each group) E. Apoptosis level was assessed with Annexin-V (PS externalization) by flow cytometry analysis in WT, Parkin -/-, PINK-/-, WT treated with H O or Parkin-/- treated with H O or PINK-/- treated with H O (.5 or mm). Graph indicates the percentage of Annexin V positive cell in the total cell population (p<., p<.5, NS no significance, n=3 for each group) 6

7 Appendix Figure S. Western blot analysis of p53, JNK, LC3I/II in NAC treatment. A. In vivo treatment of WT vs mice with NAC (5mg/kg I.P. for hour) followed by CellRox detection of ROS and flow cytometry. (p<.) B. ΔΨm (TMRM) and platelet apoptosis (Annexin-V) were measured by flow cytometry analysis in WT, and with NAC as described in Panel A. (p<.5) C. Western blot analysis of JNK, LC3I/II in, and platelets treated with NAC. GAPDH was used as the loading control. Graph indicate quantification of phosphorylation of p53 (S5), phosphorylation of JNK and LC3II (p<., p<.5 vs. ). GAPDH served as a loading control. D. In vivo treatment of WT vs PINK-/- mice with NAC (5mg/kg I.P. for hour) followed by CellRox detection of ROS and flow cytometry. (p<.) E. WT vs PINK-/- mice platelet apoptosis (Annexin-V) were measured by flow cytometry analysis in WT, and with NAC as described in Panel A. (p<.5) Appendix Figure S3. Assessment of purity of prepared platelet preparation To assess for washed platelet contamination by different blood cells, we used the monocyte/macrophagy marker CD4 and the erythrocyte marker CD35a for Western blotting. Shown is a representative Western analysis. 7

8 Appendix Table S: Clinical characteristics of healthy control and patients with type Healthy control T n=8 n=66 Age (years ± SD) 4.5± ±.6 Gender (Males/Females) 8/ 4/6 BMI (kg/m ± SD).8±. 34.±8.6 Blood glucose (mg/dl ± SD) 3.8± ±66.8 HbAc (% ± SD) 5.±.3 7.±.8 Systolic blood pressure (mmhg ± SD) 4.± ±4.7 Diastolic blood pressure (mmhg ± SD) 77.4± ±.7 Hypertension (%) /8 (%) 47/66 (7.%) CAD (%) /8 (%) 7/66 (5.8%) Medications (%) Aspirin /8 (%) 3/66 (48.5%) Clopidogrel /8 (%) 3/66 (4.5%) Warfarin /8 (%) 4/66 (6.%) Statin /8 (%) 4/66 (6.%) Beta-blocker /8 (%) 3/66 (45.5%) Angiotensin converting enzyme inhibitor /8 (%) 6/66 (39.4%) Angiotensin receptor blocker /8 (%) 4/66 (.%) Insulin /8 (%) 3/66 (45.5%) Metformin /8 (%) /66 (3.8%) Other anti-diabetic drugs /8 (%) 8/66 (.%) Diuretic /8 (%) /66 (3.8%) 8

9 Appendix Table S: Antibody information Company Target Cat# Lot# Clone # Species Titer Cell Signaling patk 46S 6 Rabbit : AKT 967 Rabbit : ATG3 35P Rabbit : ATG DB Rabbit : ATG 48 D88H Rabbit : Beclin 5495s D4C5 Rabbit : BNIP3L/NIX 396S Rabbit : Cleaved Caspase3 966s 43 D75 Rabbit : Cytochrome C 47S 6 Rabbit : GAPDH 8L 8 Rabbit : pjnk 467S 7 Rabbit : JNK 958S 9 Rabbit : LAMP 99S Rabbit : (WB) :3 (IF) PINK 6946P D8G3 Rabbit : Parkin 4S 4 Mouse : pp53(s5) 984 Rabbit : (WB) :5 (IF) P53 54 Mouse : pmtor(s448) 97S Rabbit : mtor 97S 6 Rabbit : pulk(s37) 753S Rabbit :5 ULK 4773S Rabbit :5 Ubiqitin 3936S 9 Mouse : ps6 5364S 6 Rabbit : S6 7 Rabbit : pp7s6k 95L Rabbit : 9

10 p7s6k 9L 4 Rabbit : Abcam LC3 Ab48394 Rabbit :5 Parkin Ab5954 Rabbit :5 CD4 Ab33335 Rabbit : Glycophorin Ab94 Rabbit : Malondialdehyde Ab7647 Rabbit : 4 Hydroxynonenal Ab46545 Rabbit : Cosmo LC3 LC3 73 Mouse : (IF) Santa cruze Cox4 SC-6936 G- goat : (IF) Tubulin Sigma Β-actin A536 AC-74 mouse :5

11 Appendix Figure S A kda B NT 37 4-HNE 37 DNP 5 GAPDH ubiquitin 3-NT 4-HNE DNP ubiquitin

12 Appendix Figure S µm µm

13 Appendix Figure S3 A CoxIV LC3 LAMP Merged µm Colocalization of COxIV, LC3 and LAMP (fold change vs.) B Parkin C I II LC3 CoxIV D Cyto CoxIV LC3 Parkin Merged µm Mito GAPDH Colocalization of COxIV, LC3 and Parkin (fold change vs.) 5nm Red: LC3 Black: Parkin 5 4 3

Appendix Figure S4 A 7 8 9 9 3 4 5 6 patient # Bnip3L/Nix Parkin I IILC3I/II

14 Appendix Figure S4 A patient # Bnip3L/Nix Parkin I IILC3I/II actin B Fold change vs. 5 5 Bnip3L/NIX Parkin LC3 II Healthy Control()

15 Appendix Figure S5 A Relative expression (/8sRNA) NADH dehydrogenase subunit (ND) s RNA B Relative expression (/8sRNA) NADH dehydrogenase subunit (ND).5.5 WT

16 Appendix Figure S6 A patient # pulk(757) Fold change vs pulk(757)/ulk Healthy Control() Ulk GAPDH B patient # pp7s6k ps6 actin 6 pp7s6k.5 ps6 Fold change vs Healthy Control()

17 Appendix Figure S7 A PFT-α - (μm) C HO pp53(ser5) p53 LC3I/II GAPDH B Fold change vs. pp Mitotracker CoxIV LC3 Merged P=.58 HO 6 3 LC3 II ns HO/PFT-α(μM) μm HO HO PFT-α

Appendix Figure S8 A CCCP HG HG CCCP C % of TMRM-ve platelets CoxIV LC3 Parkin Merged µm 35 3 5 5 5 HG.

18 Appendix Figure S8 A CCCP HG HG CCCP C % of TMRM-ve platelets CoxIV LC3 Parkin Merged µm HG. ns HG/HO HG/HO/3MA D B Colocalization of CoxIV, LC3 and Parkin (fold change vs. ) % of Annexin V+ve platelets SO HG CCCP SO HG/HO CCCP HG/HO/3MA HG

19 Appendix Figure S spautin- pp53(s5) I II LC3I/II GAPDH Fold change vs pp LC3II +spautin-

20 Appendix Figure S SO CCCP Aggregation (%of light transmission) A SO CCCP B C Arbitrary unit(a.u) SO CCCP Collagen LC3II LC3I/II I II GAPDH SO CCCP SO+Collagen CCCP+Collagen

21 LC3II (fold change vs WT) Appendix Figure S A WT WT/HO WT None HO PINK-/- PINK-/- None HO PINK-/- /HO I II LC3I/II GAPDH B % of TMRM-ve platelets None ns HO C % of Annexin V+ve platelets 3 WT PINK -/- None HO D E % of TMRM-ve platelets None HO.5 HO (mm) % of Annexin V+ve platelets p=.7 None HO.5 HO (mm) WT Parkin -/- PINK -/-

22 Appendix Figure S A ROS (MFI) B unstaining WT (n=) (n=3) _nac (n=3) % of TMRM-ve platelets % of Annexin V+ve platelets WT (n=5) (n=4) _nac (n=3) C /NAC pjnk I II LC3I/II GAPDH Fold change vs pjnk p=.56 LC3II NAC D ROS (MFI) E unstaining WT PINK-/- PINK-/- _nac % of Annexin V+ve platelets 5 WT PINK-/- PINK-/- _nac

23 Appendix Figure S3 Washed platelet Buffy coat CD4:platelet CD4:monocyte CD35a:erythrocyte

Inducing mitophagy in diabetic platelets protects against severe oxidative stress

Research Article Inducing mitophagy in diabetic platelets protects against severe oxidative stress Seung Hee Lee 1, Jing Du 1, Jeremiah Stitham 1, Gourg Atteya 1, Suho Lee 2, Yaozu Xiang 1, Dandan Wang