Thrombin, collagen, arachi-donic acid (AA), luciferin-luciferase, U46619, phorbol 12,13-dibutyrate (PDBu), nitroglycerin (NTG), heparin, prostaglandin E₁ (PGE₁), 5,5-dimethyl-1-pyrroline N-oxide (DMPO), SQ22536, 1H-[1,2,4]oxadiazolo[4,3-a]quinox-alin-1-one (ODQ), and bovine serum albumin (BSA) were purchased from Sigma-Aldrich; Merck KGaA (Darmstadt, Germany). Fura-2AM was obtained from Molecular Probes; Thermo Fisher Scientific, Inc. (Waltham, MA, USA). Anti-phosphorylated (p)-p38 mitogen-activated protein kinase (MAPK) Thr180/Tyr182 monoclonal antibodies (mAbs; Cat. no. 9211) were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Anti-p38 MAPK (Cat. no. 9217), anti-p-c-Jun N-terminal kinase (JNK; Thr183/Tyr185; Cat. no. 9251), and anti-p44/42 extracellular signal-regulated kinase (ERK) mAbs (Cat. no. 9107), as well as anti-phospholipase Cγ2 (PLCγ2; Cat. no. 3872), anti-p-(Tyr759) PLCγ2 (Cat. no. 3874), anti-p-(Ser) protein kinase C (PKC) substrate (pleckstrin; p-p47; Cat. no. 2261), anti-JNK (Cat. no. 9252), and anti-p-p44/p42 ERK (Thr202/Tyr204) polyclonal antibodies (pAbs; Cat. no. 9101) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Anti-p-protein kinase B (Akt) (Ser473; Cat. no. 9271) and anti-Akt mAbs (Cat. no. 2920) were purchased from Biovision (Mountain View, CA, USA). An anti-pleckstrin (p47) pAb (Cat. no. GTX17020) was purchased from Gene Tex (Irvine, CA, USA). A Hybond-P polyvinylidene fluoride (PVDF) membrane, an enhanced chemiluminescence western blotting detection reagent, horseradish peroxidase (HRP)-conjugated donkey anti-rabbit immunoglobulin G (IgG; Cat. no. RPN4301) and sheep anti-mouse IgG (Cat. no. RPN4201) were purchased from GE Healthcare Life Sciences (Little Chalfont, UK). A fluorescein isothiocyanate (FITC)-conjugated anti-human CD42P (P-selectin) mAb (Cat. no. 304904) was obtained from Bio Legend, Inc. (San Diego, CA, USA).

Ammonium acetate (1.93 g, 25 mM), 4-methoxy benzaldehyde (1.02 g, 7.5 mM) and glacial acetic acid (25 ml) were added to a degassed mixture of di-pyridin-2-yl-metha-none (0.9 g, 5 mM), and the mixture was placed in an oil bath maintained at 120°C under a nitrogen atmosphere for 18 h. The reaction mixture was then cooled to room temperature, poured into ice cold water, and extracted with chloroform (3×70 ml). The organic layer was dried over anhydrous sodium sulfate, and then filtered. The residue was chromatographed through silica gel (ethylacetate/hexane = 1:3) to yield a yellow solid. The characteristics of this solid were as follows: Melting point: 105–110°C; ¹H nuclear magnetic resonance (NMR; 400 MHz, CDCl₃): δ 8.72–8.70 (d, 1H, J=8 Hz), 8.65–8.64 (d, 1H, J=4 Hz), 8.27–8.25 (d, 1H, J=8 Hz), 8.2–8.19 (d, 1H, J=8 Hz), 7.78–7.73 (t, 3H, J=10 Hz), 7.13–7.07 (m, 3H, J=8 Hz), 6.96–6.92 (t, 1H, J=8 Hz), 6.67–6.64 (t, 1H, J=6 Hz), 3.9 (s, 3H); ¹³C NMR (400 MHz, CDCl₃) 160.0, 155.1, 148.9, 138.0, 136.1, 130.1, 129.9, 129.7, 122.5, 121.7, 121.5, 120.8, 120.3, 119.8, 114.4, 113.6, 55.3; ultraviolet-visible spectrophotometry (UV-Vis; λ_abs, nm): 380, 321, 300, 238; electrospray ionization-mass spectrometry (ESI-MS; m/z): 301 (M⁺).

[Ir(Cp*)(Cl)₂]₂ (0.16 g, 0.2 mM) in methanol (10 ml) solution was added dropwise to 10 ml methanolic solution of 1-(2-pyridyl)-3-(3-methoxy-phenyl)imidazo[1,5-a]pyridine (L) (0.12 g, 0.4 mM), and the solution was stirred at room temperature for 3 h. Subsequently, NH₄BF₄ (200 mg, 0.60 mM) was added to the initial pale yellow solution, changing the color to orange. After 24 h, the solution was evaporated and the solid obtained was filtered. The residue was washed with diethyl ether (40 ml) and dried under a vacuum. The desired product was recrystallized from the dichloromethane/hexane mixture, yielding orange micro-crystals. The characteristics of this solid were as follows: ¹H NMR (400 MHz, dimethyl sulfoxide [DMSO]-D6): δ 8.82–8.81 (d, 1H, J=4 Hz), 8.52–8.39 (m, 3H), 8.17–8.10 (m, 3H), 7.54–7.47 (m, 2H), 7.31–7.29 (d, 2H, J=8 Hz), 7.15–7.11 (t, 1H, J=8 Hz), 3.90 (s, 3H), 1.29 (s, 15H); UV-Vis (λ_abs, nm) (ε, M⁻¹ cm⁻¹): 406 (1649), 385 (2372), 367 (1854), 282 (2871), 242 (2881); ESI-MS (m/z): 664.08 [M-BF₄]⁺ (Fig. 1).

The present study was approved by the Institutional Review Board of Taipei Medical University (Taipei, Taiwan; approval no. TMU-JIRB-N201612050) and conformed to the directives of the Declaration of Helsinki. All human volunteers involved in the present study provided written informed consent. Human platelet suspensions were prepared as described previously (10). Human blood was collected from 20 healthy individuals (aged between 20 and 30 years old, 12 females and 8 males) of Taipei Medical University who had taken no drugs or other substances that would interfere with the experiment for at least 14 days prior to collection between July 2017 and August 2017. The collected blood was mixed with an acid-citrate-dextrose solution (9:1, v/v) and centrifuged at 120 × g for 10 min at 37°C to separate the platelet-rich plasma (PRP). Following centrifugation, the PRP was supplemented with 0.5 µM PGE₁ and 6.4 IU/ml heparin, incubated for 10 min at 37°C and centrifuged at 500 × g for 10 min at room temperature. The collected pellets were mixed with 5 ml Tyrode's solution, pH 7.3 [containing (mM) NaCl 11.9, KCl 2.7, MgCl₂ 2.1, NaH₂PO₄ 0.4, NaHCO₃ 11.9, and glucose 11.1]. Then, apyrase (1.0 U/ml), PGE₁ (0.5 µM) and heparin (6.4 IU/ml) were added, and the mixture was incubated for 10 min at 37°C. Following centrifugation of the suspensions at 500 × g for 10 min at room temperature, the washing procedure was repeated. The washed platelets were finally suspended in Tyrode's solution containing BSA (3.5 mg/ml). The final Ca²⁺ concentration in the Tyrode solution was 1 mM. A platelet aggregation test was performed using a Lumi-Aggregometer (Payton Associates, Scarborough, ON, Canada) as described previously (10). Various concentrations of Ir-3 or a solvent control (0.1% DMSO) were preincubated with platelet suspensions (3.6×10⁸ cells/ml) for 3 min prior to the addition of various concentrations of agonists (1 µg/ml collagen, 0.01 U/ml thrombin, 1 µM U46619 or 120 µM AA). The extent of platelet aggregation was calculated as the percentage with respect to the control (absence of Ir-3) in light transmission units when the reaction had proceeded for 6 min. For an ATP release assay, 20 µl luciferin-luciferase was added 1 min prior to the addition of the agonist, and the amount of ATP released was compared with that released by the control using a Lumi-Aggregometer.

The [Ca²⁺] concentration was measured using the calcium sensitive dye Fura-2AM, as described previously (10). In brief, citrated whole blood was centrifuged at 120 × g for 10 min at room temperature, and the supernatant was collected and incubated with 5 µM Fura-2AM for 1 h at 37°C. The Fura-2AM-preincubated platelets were washed with Tyrode's solution and treated with Ir-3 in the presence of 1 mM CaCl₂, and then stimulated with collagen (1 µg/ml). Fura-2 fluorescence was measured using excitation wavelengths of 340 and 380 nm and emission at 510 nm with a spectrofluo-rometer (Hitachi FL Spectrophotometer F-4500; Hitachi, Ltd., Tokyo, Japan).

Washed platelets (3.6×10⁸ cells/ml) were preincubated with 20–100 µM Ir-3 or the solvent control (0.1% DMSO) for 20 min at 37°C. Following incubation, supernatant (10 µl) was added to a Fuji Dri-Chem slide lactate dehydrogenase (LDH)-PIII (Fujifilm Holdings Corporation, Tokyo, Japan). The absorbance of the supernatant was measured at 540 nm using a UV-Vis spectrophotometer (UV-160; Shimadzu Corporation, Kyoto, Japan). A maximal value of LDH was recorded in platelets lysed with Triton.

Platelet surface P-selectin expression was examined using flow cytometric analysis. Washed platelets were prepared as aforementioned. Aliquots of platelet suspensions (3.6×10⁸ cells/ml) were treated with either the solvent control (0.1% DMSO) or Ir-3 (10 and 20 µM), and FITC-conjugated P-selectin (2 µg/ml) mAbs were then added and incubated for 3 min at 37°C. This was followed by the addition of collagen (1 µg/ml) to trigger platelet activation, for 1 min at 37°C. The suspensions were then assayed for fluorescein-labeled platelets using a flow cytometer (FACScan System; BD Biosciences, San Jose, CA, USA) with FACSuite™ software (version 1.0.5.3841; BD Biosciences). Data were collected from 50,000 platelets per experimental group, and the platelets were identified on the basis of their characteristic forward and orthogonal light-scattering profiles. All experiments were repeated at least three times to ensure reproducibility.

Washed platelets (1.2×10⁹ cells/ml) were preincubated with Ir-3 (10 and 20 µM) or the solvent control (0.1% DMSO) for 3 min at 37°C, and 1 µg/ml collagen was added to trigger platelet activation for 5 min at 37°C. The reaction was then stopped, and the platelets were immediately resuspended in 200 µl lysis buffer [containing (mM) 50 HEPES, 5 EDTA, 50 NaCl and 1% Triton X-100]. Proteins were quantified using an ELISA reader at 570 nm. Samples containing 80 µg of protein were separated through 12% sodium dodecyl sulfate gel electrophoresis, and the proteins were electrotrans-ferred to PVDF membranes using a Bio-Rad semi-dry transfer unit (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The blots were then blocked with Tris-buffered saline in Tween-20 (TBST; 10 mM Tris-base, 100 mM NaCl, and 0.01% Tween-20) containing 5% BSA for 1 h at room temperature, and probed with the aforementioned primary antibodies (diluted 1:1,000 in TBST) for 2 h at 4°C. The membranes were incubated with HRP-conjugated anti-mouse IgG for anti-ERK, anti-p38 and anti-Akt or anti-rabbit IgG for anti-phosphorylated MAPKs, anti-p-Akt, anti-p-PLCγ2, anti-PLCγ2, anti-p-PKC substrate and anti-JNK (diluted 1:3,000 in TBST) for 1 h at 4°C. An enhanced chemiluminescence system was used to detect immunoreactive bands, and their optical density was quantified using Bio-profil Biolight software (version V2000.01; Vilber Lourmat, Marne-la-Vallée, France).

Electron spin resonance spectrometry was performed on a Bruker EMX ESR spectrometer (Bruker Corporation, Billerica, MA, USA) as described previously (11). Platelet suspensions (3.6×10⁸ cells/ml) or Fenton reaction solution (50 µM FeSO₄+2 mM H₂O₂) were preincu-bated with 0.1% DMSO or Ir-3 (10 and 20 µM) for 3 min at room temperature, with or without the addition of 1 µg/ml collagen. Following incubation of the suspensions for 5 min, 100 µM DMPO was added prior to the execution of ESR spectrometry. The ESR spectra were recorded using a quartz flat cell designed for aqueous solutions. The spectrometer was operated at a power of 20 mW, frequency of 9.78 GHz, scan range of 100 G and receiver gain of 5×10⁴. The modulation amplitude was 1 G, the time constant was 164 ms, and scanning was performed for 42 sec, with the spectra being the sum of three scans.

The experimental results are expressed as the mean ± standard error of the mean, and are accompanied by the number of observations (n). The n values refer to the number of experiments, and each experiment was conducted using different blood donors. The between-group differences in the experiments were assessed through one-way analysis of variance (ANOVA). When the ANOVA indicated significant differences among the group means, the groups were compared using the Student-Newman-Keuls method. Statistical analyses were performed using SAS (version 9.2; SAS Institute, Inc., Cary, NC, USA). P<0.05 was considered to indicate a statistically significant difference.

Ir-3 (5–20 µM; Fig. 2) inhibited platelet aggregation in collagen (1 µg/ml)-stimulated human platelets in a concentration-dependent manner. Ir-3 (20–100 µM) demonstrated moderate activity against platelet aggregation in platelets stimulated by 120 µM AA. Furthermore, Ir-3 (100–500 µM) had relatively weak activity against 0.01 U/ml thrombin or 1 µM U46619, a prostaglandin endoperoxide stimulant, indicating that Ir-3 had more potent inhibitory activity against collagen stimulation compared with other agonists (Fig. 2B). The solvent control, 0.1% DMSO, did not affect platelet aggregation (Fig. 2A). Furthermore, aspirin (20, 50 and 100 µM) inhibited platelet aggregation stimulated by 1 µg/ml collagen in a concentration-dependent manner, with a half maximal inhibitory concentration of ~50 µM (n=3; data not shown). Therefore, Ir-3 is ~5 times more potent than aspirin at inhibiting collagen-stimulated platelet aggregation. In the following experiments, 1 µg/ml collagen was used as an agonist for investigating potential inhibitory mechanisms of Ir-3 in human platelets.

The activation of platelets releases the granular contents, including adenosine diphosphate (ADP)/ATP and Ca⁺² from the dense granules, and surface P-selectin from the α-granules, to the external environment where they are involved in substantial platelet aggregation. In the present study, Ir-3 (10 and 20 µM) inhibited the ATP release reaction (10 µM, 33.2±1.1%; 20 µM, 67.8±3.7%; Fig. 3A) and relative [Ca²⁺]i mobilization (resting control, 78.4±14.3 nM; collagen-stimulated, 438.9±82.6 nM; 10 µM, 221.9±33.3 nM; 20 µM, 119.1±23.8 nM; n=3, Fig. 3B) in platelets stimulated by 1 µg/ml collagen. The corresponding statistical data are presented in the right panels of Fig. 3. In quiescent platelets, P-selectin is located on the inner wall of the α-granules. Platelet activation exposes the inner walls of the granules to the outside of the cell (12). Ir-3 treatment significantly reduced collagen-induced surface P-selectin expression, as demonstrated by the corresponding statistical data in the right panel (resting control, 61.7±11.5; collagen-activated, 655.7±137.3; 10 µM, 396.7±128.8; 20 µM, 181.7±63.8; n=3; Fig. 3C).

The aggregation curves of platelets pre-incubated with 100 µM Ir-3 for 10 min and washed two times with Tyrode solution were not significantly different from those of platelets pre-incubated with the solvent control (0.1% DMSO) under equivalent conditions (Fig. 4A), preliminarily indicating that the effects of Ir-3 on platelet aggregation are reversible and non-cytotoxic. Furthermore, the LDH results revealed that Ir-3 (20, 50, and 100 µM) incubated with platelets for 20 min did not significantly increase LDH activity or exert cytotoxic effects on platelets (Fig. 4B), demonstrating that Ir-3 does not affect platelet permeability or induce platelet cytolysis. Furthermore, 100 µM SQ22536, an adenylate cyclase inhibitor, and 10 µM ODQ, a guanylate cyclase inhibitor, significantly reversed the inhibition of collagen-induced platelet aggregation mediated by 1 µM PGE₁ or 10 µM NTG (Fig. 4C and D). Neither SQ22536 nor ODQ significantly reversed the inhibition of collagen-induced platelet aggregation mediated by 20 µM Ir-3 (Fig. 4C and D), indicating that the mechanisms of Ir-3-mediated inhibition of platelet aggregation are independent of increasing cyclic nucleotide formation.

PLCs hydrolyze phosphatidylinositol 4,5-bisphosphate to generate the secondary messengers inositol 1,4,5-trisphosphate (IP₃) and diacylglycerol (DAG). IP₃ triggers relative [Ca²⁺] mobilization and DAG activates PKC, yielding a ~47-kDa protein that is predominantly phosphorylated (p47 protein; pleckstrin) and leads to the ATP release reaction (13). Fig. 3A and B illustrate the inhibitory effects of Ir-3 against ATP release and relative [Ca²⁺] mobilization. The influence of Ir-3 on the phosphorylation of the PLCγ2-PKC signaling cascade was then investigated further. At 10 and 20 µM, Ir-3 significantly reduced PLCγ2 phosphorylation and PKC activation (pleckstrin phosphorylation) in collagen-stimulated platelets compared with the DMSO control (Fig. 5A and B). However, Ir-3 had no significant effects on platelet aggregation stimulated by 150 nM PDBu, a PKC activator (Fig. 5C), indicating that Ir-3 does not directly disturb PKC activation but may interfere with upstream regulators of PKC, including PLCγ2. Akt is a serine/threonine-specific protein kinase that serves a key function in multiple cellular processes, including platelet activation, cell proliferation, apoptosis and cell migration (14). Ir-3 (10 and 20 µM) significantly inhibited collagen-induced Akt phosphorylation (Fig. 5D) compared with the DMSO control, demonstrating the involvement of Akt signaling pathway inhibition in Ir-3-mediated inhibition of platelet activation.

To investigate the inhibitory mechanisms of Ir-3 in platelet activation, several signaling molecules of the MAPK phosphorylation pathway were evaluated. MAPKs, including p38 MAPK, ERKs, and JNKs, regulate major cellular responses in eukaryotic organisms and contribute to cell proliferation, migration, differentiation, and apoptosis (15). ERKs, JNK1, and p38 MAPK have been identified in platelets (16). Although collagen-induced p38 MAPK and ERK phosphorylation was unaffected by Ir-3, JNK1 phosphorylation was suppressed by 10 and 20 µM Ir-3 in a concentration-dependent manner (Fig. 6). These results suggest that only JNK1 may be involved in the antiplatelet activity of Ir-3.

An ESR signal indicative of OH· formation was observed in collagen-stimulated platelet suspensions and Fenton reaction solution (cell-free system; Fig. 7A and B). A typical OH· signal (a^N =a^H = 14.8 G) and a long-lived g=2.005 radical detectable using the spin trap DMPO were observed in collagen-stimulated platelets, whereas this signal was not detected in resting platelets (Fig. 7A and B). Treatment with 10 and 20 µM Ir-3 did not notably diminish the OH· signals in collagen-stimulated platelet suspensions or Fenton reaction solution (Fig. 7), suggesting that the Ir-3-mediated inhibition of platelet activation may not be mediated through the reduction of free radical formation.