Hydrogen Sulfide Inhibits Human Platelet Aggregation In Vitro in Part by Interfering Gap Junction Channels: Effects of ACS14, a Hydrogen Sulfide-releasing Aspirin  Lin Gao, Chun Cheng, Anna Sparatore, Huili Zhang, Changqian Wang  Heart, Lung and Circulation  Volume 24, Issue 1, Pages 77-85 (January 2015) DOI: 10.1016/j.hlc.2014.05.019 Copyright © 2014 Australian and New Zealand Society of Cardiac and Thoracic Surgeons (ANZSCTS) and the Cardiac Society of Australia and New Zealand (CSANZ) Terms and Conditions

Figure 1 Concentration- and time-related effects of ACS14 on agonist-induced platelet aggregation. (A, B, E, F) after incubation of human platelets with ACS14 or aspirin at concentrations ranging from 10μM to 200μM for 30minutes, ADP (2.5μM), thrombin (0.1U/mL), collagen (2.5μg/ml) or AA (0.5mM) stimulated platelet aggregation was assayed as described in Materials and Methods. (C, D) after incubation of human platelets with ACS14 (100μM) or aspirin (100μM) for up to 120minutes, ADP (2.5μM) or thrombin (0.1U/mL) stimulated platelet aggregation was assayed. * P<0.05, compared with control; # P<0.05, compared with aspirin at the same concentration. Heart, Lung and Circulation 2015 24, 77-85DOI: (10.1016/j.hlc.2014.05.019) Copyright © 2014 Australian and New Zealand Society of Cardiac and Thoracic Surgeons (ANZSCTS) and the Cardiac Society of Australia and New Zealand (CSANZ) Terms and Conditions

Figure 2 Effect of ACS14 on platelet αIIbβ3 integrin binding and P-selectin expression. In washed human platelets, ACS14 (100μM for 30minutes) resulted in a significant reduction in αIIbβ3 integrin binding on stimulation with ADP (2.5μM) (A) or thrombin (0.1U/mL) (B), as detected by PAC-1 antibody. Meanwhile, surface expression of P-selectin on stimulation with ADP (2.5μM) (D) or thrombin (0.1U/mL) (E) was also significantly inhibited by ACS14 (100μM for 30minutes). Histograms or blots (C, F) are representative for 5 to 6 independent experiments. * P<0.05, compared with aspirin at the same concentration. Heart, Lung and Circulation 2015 24, 77-85DOI: (10.1016/j.hlc.2014.05.019) Copyright © 2014 Australian and New Zealand Society of Cardiac and Thoracic Surgeons (ANZSCTS) and the Cardiac Society of Australia and New Zealand (CSANZ) Terms and Conditions

Figure 3 Effect of NaHS on agonist-induced platelet aggregation. After incubation of human platelets with NaHS at concentrations ranging from 10μM to 200μM for 30minutes, ADP (2.5μM), thrombin (0.1U/mL), AA (0.5mM) or collagen (2.5μg/ml) stimulated platelet aggregation was assayed. * P<0.05, compared with control. Heart, Lung and Circulation 2015 24, 77-85DOI: (10.1016/j.hlc.2014.05.019) Copyright © 2014 Australian and New Zealand Society of Cardiac and Thoracic Surgeons (ANZSCTS) and the Cardiac Society of Australia and New Zealand (CSANZ) Terms and Conditions

Figure 4 Effect of NaHS on platelet αIIbβ3 integrin binding and P-selectin expression. In washed human platelets, NaHS (100μM for 30minutes) resulted in a significant reduction in αIIbβ3 integrin binding (A, B) and surface expression of P-selectin (D, E) on stimulation with ADP (2.5) (A) or thrombin (0.1U/mL). Histograms or blots (C, F) are representative for 5 to 6 independent experiments. * P<0.05, compared with control. Heart, Lung and Circulation 2015 24, 77-85DOI: (10.1016/j.hlc.2014.05.019) Copyright © 2014 Australian and New Zealand Society of Cardiac and Thoracic Surgeons (ANZSCTS) and the Cardiac Society of Australia and New Zealand (CSANZ) Terms and Conditions

Figure 5 Presence of gap junction–like structures between activated platelet membranes was analysed with transmission electron microscopy. Arrows indicate gap junction-like structures. Heart, Lung and Circulation 2015 24, 77-85DOI: (10.1016/j.hlc.2014.05.019) Copyright © 2014 Australian and New Zealand Society of Cardiac and Thoracic Surgeons (ANZSCTS) and the Cardiac Society of Australia and New Zealand (CSANZ) Terms and Conditions

Figure 6 Effect of rotigaptide (R), a gap junction channel modifier on the anti-platelet property of ACS14 or NaHS. A, B) Human platelets were pretreated with rotigaptide (10nM) for 1 hour and then incubated with ACS14 (100μM) (A) or NaHS (100μM) (B) for 30minutes. Afterward, platelet aggregation induced by ADP (2.5μM) or thrombin (0.1U/mL) was assayed. C-H) Washed human platelets were pretreated with rotigaptide (R, 10nM) for 1 hour and then incubated with ACS14 (100μM) (C, E) or NaHS (100μM) (G, H) for 30minutes. Afterward, platelet αIIbβ3 integrin binding activity and surface P-selectin expression induced by ADP (2.5μM) or thrombin (0.1U/mL) was assayed. Histograms or blots (D, F, I) are representative for 5 to 6 independent experiments. * P<0.05, compared with control; # P<0.05, compared with platelets pretreated with rotigaptide. Heart, Lung and Circulation 2015 24, 77-85DOI: (10.1016/j.hlc.2014.05.019) Copyright © 2014 Australian and New Zealand Society of Cardiac and Thoracic Surgeons (ANZSCTS) and the Cardiac Society of Australia and New Zealand (CSANZ) Terms and Conditions