Ir-6: A Novel Iridium (III) Organometallic Derivative for Inhibition of Human Platelet Activation

Figures

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  Figure 1. Comparison among the levels of Ir-6-mediated inhibition of platelet aggregation induced by agonists in washed human platelets. (a) Synthesis of complex (Ir-6) [Ir(Cp∗)(L)Cl]BF4. (b) Platelets (3.6 × 108 cells/mL) were preincubated with the solvent control (0.1% DMSO) or Ir-6 (5–500 μM) and subsequently treated with collagen (1 μg/mL), arachidonic acid (AA; 120 μM), thrombin (0.01 U/mL), or U46619 (1 μM) to stimulate platelet aggregation. (c) Concentration–response histograms of Ir-6 representing the levels of inhibition of platelet aggregation stimulated by various agonists. Data are presented as the mean ± standard the error of the mean (n=4).

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  Figure 2. Effect of Ir-6 on ATP-release reaction, relative [Ca2+]i mobilization, and surface FITC-P-selectin expression in washed human platelets. Platelets (3.6 × 108 cells/mL) were preincubated with the solvent control (0.1% DMSO) or Ir-6 (10 and 20 µM). Subsequently, collagen (1 μg/mL) was added to trigger either (a) the ATP-release reaction (AU; arbitrary unit) or (b) relative [Ca2+]i mobilization (nM) in calcium-free Tyrode's solution (A) or Tyrode's solution (B). (c) Washed platelets (3.6 × 108/mL) were preincubated with the solvent control (0.1% DMSO) or Ir-6 (10 and 20 µM) and FITC-P-selectin (2 µg/mL) for 3 min and then stimulated using collagen (1 μg/mL). The corresponding statistical data are shown in the right panel of each Figures 2(a)–2(c). Data are presented as the mean ± standard error of the mean (n=4). ∗P < 0.01 and ∗∗∗P < 0.001, compared with the DMSO-treated group (a) or resting control (b and c); #P < 0.05, ##P < 0.01, and ###P < 0.001, compared with the DMSO-treated group (b and c).

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  Figure 3. Effect of Ir-6 on cyclic nucleotide formation, LDH release, and cytotoxicity in human platelets. (a) Washed platelets (3.6 × 108 cells/mL) were preincubated with 1 μM PGE1, 10 μM NTG, or 20 µM Ir-6 with or without 100 μM SQ22536 or 10 μM ODQ and were subsequently treated with collagen (1 μg/mL) to induce platelet aggregation. (b) Washed platelets (3.6 × 108 cells/mL) were preincubated with the solvent control (0.1% DMSO) or with various concentrations of Ir-6 (20, 50, and 100 µM) for 20 min, and a 10 µL aliquot of the supernatant was deposited on a Fuji Dri-Chem slide LDH-PIII. (c) For other experiment, washed platelets were preincubated with the solvent control (0.1% DMSO) or Ir-6 (100 μM) for 10 min and subsequently washed two times with Tyrode's solution. Collagen (1 μg/mL) was then added to trigger platelet aggregation. Data are presented as the mean ± standard error of the mean (n=4). Profiles in (a) and (c) represent four independent experiments.

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  Figure 4. Effects of Ir-6 on PLCγ2, PKC, and Akt activation in human platelets. Washed platelets were preincubated with the solvent control (0.1% DMSO), LY294002 (10 µM), or Ir-6 (10 or 20 µM). Subsequently, either collagen (1 μg/mL) or PDBu (150 nM) was added to trigger (a) platelet aggregation, (b) PLCγ2 phosphorylation, and (c) PKC activation (pleckstrin phosphorylation), or (d) Akt activation. Platelets were collected, and their subcellular extracts were analyzed to determine the levels of phosphorylation of (b) PLCγ2, (c) PKC, and (d) Akt. Data are presented as the mean ± standard error of the mean (n=4). ∗P < 0.05 and ∗∗∗P < 0.001, compared with the resting control; #P < 0.05 and ###P < 0.001, compared with the DMSO-treated group. The profiles in (a) and (d, left panel) are representative of four independent experiments.

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  Figure 5. Inhibitory effects of Ir-6 on the phosphorylation of p38 MAPK, ERK2, and JNK1 in human platelets. The washed platelets (1.2 × 109 cells/mL) were preincubated with the solvent control (0.1% DMSO), SB203580 (10 µM), PD98059 (20 µM), SP600125 (10 µM), or Ir-6 (10 or 20 µM); subsequently, collagen (1 μg/mL) was added to trigger platelet activation. The platelets were collected, and their subcellular extracts were then analyzed to determine the levels of phosphorylation of (a) p38 MAPK, (b) ERK2, and (c) JNK1. Data are presented as the mean ± standard error of the mean (n=4). ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001, compared with the resting control; #P < 0.05, ##P < 0.01, and ###P < 0.001, compared with the DMSO-treated group. The profiles of left panels are representative of four independent experiments.

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  Figure 6. Regulatory activities of Ir-6 on OH· formation in either platelet suspensions or Fenton reaction solution and determination of bleeding time in experimental mice. (a) Washed platelets or (b) the Fenton reaction solution was preincubated with (A) Tyrode's solution (resting control), (B) 0.1% DMSO, (C) 10 µM Ir-6, or (D) 20 µM Ir-6. Collagen (1 µg/mL) was then added into platelet suspensions for the ESR experiments as described in Materials and Methods. Profiles in (a) and (b) are representative of four independent experiments, and an asterisk (∗) indicates OH· formation. (c) Mouse tails were transected to measure the bleeding time after 30 min of intraperitoneal administration of either 0.1% DMSO, 1.0 mg/kg Ir-6, or 2.0 mg/kg Ir-6 (all in 50 μL). Data are presented as the mean ± standard error of the mean. n=8. ∗∗P < 0.01, compared with the DMSO (0.1%)-treated group.