A novel ruthenium (II)-derived organometallic compound, TQ-6, potently inhibits platelet aggregation: Ex vivo and in vivo studies

Figures

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  Figure 1. Synthesis of TQ-6 and its inhibitory activity against agonist-induced platelet aggregation in washed human platelets. (A) Synthetic process of TQ-6. (B) Washed human platelets (3.6 × 108 cells/ml) were preincubated with the solvent control (0.5% DMSO), TQ-6 (0.1–100 μM), or aspirin (20–100 μM) and subsequently treated with 1 μg/ml collagen, 1 μM U46619, and 0.01 U/ml thrombin to stimulate platelet aggregation. (C) Concentration-inhibition histograms of TQ-6 and aspirin stimulated by various concentrations of agonists (1 and 2 μg/ml collagen; 0.01 and 0.02 U/ml thrombin; 0.5 and 1 μM U46619; 10 ng/ml convulxin) were presented. Data are presented as means ± standard errors of the means (n = 4).

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  Figure 2. Effects of TQ-6 on cytotoxicity, lactate dehydrogenase (LDH) release, ATP-release reaction, relative [Ca2+]i mobilization, and surface FITC-P-selectin expression in human platelets. (A) Washed platelets (3.6 × 108 cells/ml) were preincubated with the solvent control (0.5% DMSO) or TQ-6 (100 μM) for 10 min, and subsequently washed two times with Tyrode’s solution, followed by the addition of collagen (1 μg/ml) to trigger platelet aggregation. (B) LDH experiment: platelets were preincubated with TQ-6 (20, 50, and 100 µΜ) for 10 min, and a 10-µl aliquot of supernatant was deposited on a Fuji Dri-Chem slide LDH-PIII as described in “Materials and Methods”. Moreover, washed platelets (3.6 × 108 cells/ml) were preincubated with TQ-6 (0.3 and 0.5 µM) or the solvent control (0.5% DMSO), and 1 μg/ml collagen was then added to stimulate either (C) ATP-release reaction (AU; arbitrary unit) or (D) relative [Ca2+]i mobilization (ratio 340/380 nm). (E) Washed platelets (3.6 × 108/ml) were preincubated with TQ-6 (0.3 and 0.5 µM) or the solvent control (0.5% DMSO) and FITC-P-selectin (2 µg/ml) for 3 min, and then stimulated by collagen (1 μg/ml). Profiles in (A) are representative of four…

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  Figure 3. Effects of TQ-6 in integrin αIIbβ3 and GP VI activation as well as cyclic nucleotide formation in human platelets. (A) Washed platelets (3.6 × 108/ml) were preincubated with the (b) solvent control (0.5% DMSO) or TQ-6 (c, 0.3 µM; d, 0.5 µM) and FITC-PAC-1 (2 µg/ml) for 3 min in the (a) absence or (b–d) presence of collagen (1 μg/ml). (B) Solid line represents the fluorescence profiles of FITC-triflavin (2 µg/ml) serving only as a positive control (a); treatment with EDTA (b, 5 mM; as a negative control), TQ-6 (c, 0.3 µM; d, 0.5 µM), followed by the addition of FITC-triflavin (2 µg/ml). (C) Washed platelets were preincubated with the solvent control (0.5% DMSO) or TQ-6 (0.3 and 0.5 µM), and subsequently activated by immobilized fibrinogen (100 μg/ml). Platelets were collected, and their subcellular extracts were analyzed to determine the levels of integrin β3 phosphorylation. (D) The fluorescence profiles of (a) FITC only as a background control; washed platelets were preincubated with the (b) solvent control (0.5% DMSO) or TQ-6 (c, 0.3 µM; d, 0.5 µM) for 3 min, followed by the addition of FITC-JAQ1 (1 µg/ml). (E) For other experiments, washed platelets (3.6 × 108 cells/mL) were…

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  Figure 4. Regulatory effects of TQ-6 on the Src, Syk, and PLCγ2-PKC activation in platelets. Washed platelets were preincubated with TQ-6 or the solvent control (0.5% DMSO) and subsequently treated with either 1 μg/ml collagen, 0.01 U/ml thrombin, 1 μM U46619 or 150 nM PDBu to induce (A) Src, (B) Syk, (C) PLCγ2, (D) PKC activation (pleckstrin phosphorylation) or (E) platelet aggregation. Profiles in (A,B, E) are representative of four independent experiments. Data are presented as means ± standard errors of the means (n = 4). *p < 0.05 and **p < 0.01, compared with the 0.5% DMSO-treated group; #p < 0.05 and ##p < 0.01, compared with the collagen-treated group.

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  Figure 5. Effects of TQ-6 on ERK2, JNK1, p38 MAPK, and Akt phosphorylation in collagen-activated platelets. Washed platelets (1.2 × 109 cells/ml) were preincubated with 0.3 or 0.5 μM TQ-6 or the solvent control (0.5% DMSO) and subsequently treated with 1 μg/ml collagen to induce platelet activation. Platelets were collected, and their subcellular extracts were analyzed to determine the levels of (A) ERK2, (B) JNK1, (C) p38 MAPK, and (D) Akt phosphorylation. Data are presented as means ± standard errors of the means (n = 4). *p < 0.05, **p < 0.01, and ***p < 0.001, compared with the solvent control (0.5% DMSO) group; #p < 0.05, ##p < 0.01, and ###p < 0.001, compared with the collagen-treated group.

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  Figure 6. Protective effects of TQ-6 on OH· formation by electron spin resonance (ESR) study, closure time according to the analysis on the PFA-100 system and thrombotic platelet plug formation in the mesenteric venules of mice as well as the bleeding time in mouse tail vein. (A) Washed platelets were preincubated with Tyrode’s solution (a, resting control) or treated with (b) 0.5% DMSO or TQ-6 at (c) 0.3 µM or (d) 0.5 µM, followed by the addition of collagen (1 µg/ml) to trigger OH· formation. Profiles are representative of four independent experiments and an asterisk (*) indicates the formation of OH·. (B) Shear-induced platelet plug formation in whole blood was determined by recording the closure time of CEPI-coated membranes, as described in the “Materials and methods.” (C) For another study, mice were administered an intravenous bolus of the solvent control (0.5% DMSO) or TQ-6 (0.4 mg/kg), and the mesenteric venules were irradiated to induce microthrombus formation (occlusion time). (D) Microscopic images (400 × magnification) of 0.5% DMSO-treated controls (a, b) and the TQ-6 (0.4 mg/kg)-treated groups (c, d) were recorded at 5 (a, c) and 150 s (b, d) after irradiation. Photographs…