Sesamol, collagen (type I), prostaglandin E₁ (PGE₁), heparin, (E)-3-(4-methylphenylsulfonyl)-2-propenenitrile (BAY11-7082), 9-(tetrahydro-2-furanyl)-9H-purin-6-amine (SQ22536), N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride (H89), and 1H-124oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) were purchased from Sigma Chemical (St Louis, MO, USA); Fura 2-AM was from Molecular Probe (Eugene, OR, USA); the anti-phospho-IKKα (Ser¹⁸⁰)/IKKβ (Ser¹⁸¹) polyclonal antibody (pAb), anti-IκBα (44D4) pAb, anti-PLCγ2, anti-phospho (Tyr⁷⁵⁹) PLCγ2 monoclonal antibodies (mAbs), anti-phospho (Ser) PKC substrate (p47) pAb, and the anti-phospho-NF-κB p65 (Ser⁵³⁶) pAb were from Cell Signaling (Beverly, MA, USA); the anti-α-tubulin mAb was from NeoMarkers (Fremont, CA, USA); and the Hybond-P polyvinylidene difluoride (PVDF) membrane, enhanced chemiluminescence (ECL) Western blotting detection reagent and analysis system, horseradish peroxidase (HRP)-conjugated donkey anti-rabbit immunoglobulin G (IgG), and sheep anti-mouse IgG were from Amersham (Buckinghamshire, UK). Sesamol was dissolved in 0.5% dimethyl sulfoxide (DMSO) and stored at 4°C until used.

Human platelet suspensions were prepared as previously described 10. This study was conducted according to the guidelines laid down in the Declaration of Helsinki and all procedures involving human subjects were approved by the Institutional Review Board of Taipei Medical University, and all human volunteers provided informed consent. In brief, blood was collected from healthy human volunteers who had taken no medicine during the preceding 2 weeks, and was mixed with acid/citrate/glucose (9:1; v/v). After centrifugation at 120 g for 10 min, the supernatant (platelet-rich plasma; PRP) was supplemented with PGE₁ (0.5 μM) and heparin (6.4 IU/ml), and then incubated for 10 min at 37°C and centrifuged at 500 g for 10 min. The platelet pellets were suspended in 5 ml of 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. After centrifugation of the suspensions at 500 g for 10 min, the washing procedure was repeated. The washed platelets were finally suspended in Tyrode's solution containing bovine serum albumin (BSA) (3.5 mg/ml) and adjusted to about 4.5 × 10⁸ platelets/ml. The final concentration of Ca²⁺ in the Tyrode's solution was 1 mM.

A turbidimetric method was applied to measure platelet aggregation 10, using a Lumi-Aggregometer (Payton, Scarborough, Ontario, Canada). Platelet suspensions (3.6 × 10⁸ platelets/ml) were preincubated with various concentrations of sesamol or inhibitors for 3 min before the addition of collagen (1 μg/ml). The reaction was allowed to proceed for 6 min, and the extent of aggregation was expressed in light-transmission units.

Citrated whole blood was centrifuged at 120 g for 10 min. The PRP was incubated with Fura 2-AM (5 μM) for 1 h. Washed platelets (8 × 10⁸ platelets/ml) were then prepared as described above. Finally, the external Ca²⁺ concentration of the platelet suspensions was adjusted to 1 mM. The rise in the [Ca²⁺]i was measured using a fluorescence spectrophotometer (CAF 110, Jasco, Tokyo, Japan) with excitation wavelengths of 340 and 380 nm, and an emission wavelength of 500 nm 10.

Washed platelets (1.2 × 10⁹/ml) were preincubated with sesamol (2.5~25 μM) or various inhibitors for 3 min, followed by the addition of collagen (1 μg/ml) to trigger platelet activation. The reaction was stopped, and platelets were immediately re-suspended in 200 μl of lysis buffer (50 mM Hepes, 5 mM EDTA, 50 mM NaCl, 1% triton X-100, 10 μg/ml aprotinin, 1 mM phenylmethylsulfonylfluoride, 10 μg/ml leupeptin, 10 mM NaF, 1 mM sodium orthovanadate, 5 mM sodium pyrophosphate, and 2 mM dithiothreitol) for 1 h. Lysates were centrifuged at 5000 g for 5 min. Samples containing 80 μg of protein were separated by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) (12%); proteins were electrotransferred by a semidry transfer method (Bio-Rad, Hercules, CA). Blots were blocked with TBST (10 mM Tris-base, 100 mM NaCl, and 0.01% Tween 20) containing 5% BSA for 1 h and then probed with various primary antibodies (diluted 1:1000 in TBST). Membranes were incubated with HRP-linked anti-mouse IgG or anti-rabbit IgG (diluted 1:3000 in TBST) for 1 h. Immunoreactive bands were detected by an ECL system. The bar graph depicts the ratios of semiquantitative results obtained by scanning reactive bands and quantifying the optical density using videodensitometry (Bio-profil; Biolight Windows Application V2000.01; Vilber Lourmat, France).

In brief, washed platelets (3.6 × 10⁸/ml) were preincubated with Tyrode's solution, solvent control (0.5% DMSO), and various concentrations of sesamol (5~100 μM) for 20 min at 37°C, a 10-μl aliquot of supernatant was deposited on a Fuji Dri-Chem slide LDH-PIII (Fuji, Tokyo, Japan), and the absorbance wavelength was read at 540 nm using an ultraviolet-visible recording spectrophotometer (UV-160; Shimazu, Japan). A maximal value (MAX) of LDH was observed from sonicated platelets.

The experimental results are expressed as the means ± S.E.M. and are accompanied by the number of observations (n). Values of n refer to the number of experiments, each made with different blood donors. All experiments were assessed by an analysis of variance (ANOVA). If this analysis indicated significant differences among group means, then each group was compared using the Newman-Keuls method. p < 0.05 was considered statistically significant.

In our previous report 11, sesamol (1~5 μM) exhibited potent activity of inhibiting platelet aggregation stimulated by collagen (1 μg/ml); it also significantly inhibited platelet aggregation stimulated by arachidonic acid (AA) (60 μM) at higher concentrations (5~10 μM). In the present study, we used collagen as an agonist to further explore the inhibitory mechanisms of sesamol in platelet activation. The pleiotropic NF-κB normally exists as an inactive cytoplasmic complex, the predominant form of which is a heterodimer composed of p50 and p65 subunits tightly bound to inhibitory proteins of the IκB family 12. Once phosphorylated by the IκB kinase (IKK) complex, IκB dissociates from NF-κB subunits and is ubiquitinated and rapidly degraded by the proteasome 12. IKK phosphorylation was proposed as being a major mode for IκBα degradation, leading to NF-κB activation 12. As shown in Figure 1A, IKKβ phosphorylation significantly increased 3 min after being stimulated by collagen (1 μg/ml) in washed platelets. The compiled data of Figure 1A are shown at the bottom. Pretreatment of platelets with various concentrations of sesamol (2.5~25 μM) concentration-dependently attenuated collagen-induced IKKβ phosphorylation (Figure 1A). Based on the above results indicating that sesamol's attenuation of IKKβ phosphorylation may interfere with the IKK-IκBα cascade, we sought to further examine whether sesamol interferes with IκBα degradation. Treatment with collagen caused the rapid, time-dependent disappearance of the immunoreactive bands of IκBα (Figure 1B). The IκBα protein was markedly degraded within 10 min, and reached maximal degradation at 30 min after collagen stimulation. Sesamol (2.5~25 μM) concentration-dependently reversed the degradation of IκBα protein at 10 min after collagen stimulation (Figure 1C). These results suggest that IKKβ phosphorylation and subsequent IκBα degradation in collagen-stimulated platelets may contribute to sesamol's inhibitory actions on NF-κB signaling. In addition, the lactate dehydrogenase (LDH) study revealed that sesamol (5~100 μM) incubated with platelets for 20 min did not significantly increase LDH activity, even at a higher concentration (100 μM) (Figure 1D), indicating that sesamol did not affect platelet permeability or induce platelet cytolysis, it clearly shows that no cytotoxic effects of sesamol on platelets at these concentrations.

In our previous description 11, sesamol increased levels of both cAMP and cGMP, suggesting that increased cAMP stimulated eNOS activity and NO biosynthesis, followed by increasing cGMP formation. cAMP is the upstream regulator of the eNOS-NO-cGMP cascade in sesamol-mediated antiplatelet effects. To investigate whether sesamol-mediated inhibition of NF-κB activation was also regulated by cyclic nucleotides, especially cAMP, we used two different cyclic nucleotide inhibitors, SQ22536 (100 μM) that inhibits adenylate cyclase and ODQ (20 μM) an inhibitor of guanylate cyclase. Both inhibitors strongly reversed the sesamol (25 μM)-mediated inhibition of IKKβ phosphorylation (Figure 2A) and IκBα degradation (Figure 2B) stimulated by collagen in washed platelets. In addition, Liu et al. 13 showed that platelets express three members of the NF-κB pathway: IKK, IκB, and NF-κB p65. The present study also demonstrated that p65 phosphorylation was markedly increased, and sesamol (2.5 and 25 μM) concentration-dependently attenuated p65 phosphorylation in platelets stimulated by collagen (1 μg/ml) (Figure 2C). In the presence of SQ22536 (100 μM) and ODQ (20 μM), both inhibitors clearly reversed the sesamol (25 μM)-mediated inhibition of p65 phosphorylation (Figure 2C).

The effects of cyclic nucleotides are mediated via their respective protein kinase (i.e., PKA, a specific cAMP-dependent protein kinase), which phosphorylates substrate proteins involved in platelet inhibitory pathways 14. To investigate whether sesamol's inhibition of NF-κB was regulated by PKA, a PKA inhibitor (H89) that inhibits ATP binding to PKA catalytic subunits (PKAc) was used. As shown in the Figure 2D, H89 (5 μM) exhibited a similar effect as SQ22536 (100 μM) in reversing the sesamol-mediated inhibition of IκBα degradation.

As described previously 11, we suggest that sesamol may increase the level of cAMP, followed by inhibition of the PLCγ2-PKC cascade, thereby leading to inhibition of [Ca²⁺]i mobilization, and finally inhibition of platelet aggregation. PLC hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP₂) to generate two secondary messengers: inositol 1,4,5-trisphosphate (IP₃) and diacylglycerol (DAG) 15. DAG activates PKC, inducing 40~47-kDa protein phosphorylation. To further establish the cellular signaling events of NF-κB associated with the PLCγ2-PKC cascade in sesamol-mediated inhibition of platelet activation, an NF-κB inhibitor, BAY11-7082, which is an irreversible inhibitor of IκBα phosphorylation was used 16. The immunoblotting analysis revealed that treatment with BAY11-7082 (10 and 50 μM) concentration-dependently abolished IκBα degradation (Figure 3A) and PLCγ2 phosphorylation (Figure 3B) stimulated by collagen (1 μg/ml). When collagen was added to human platelets, a protein with an apparent molecular weight of 47 kDa (p47) was predominately phosphorylated compared to resting platelets (Figure 3C). BAY11-7082 (10 and 50 μM) also abolished p47 phosphorylation, indicating that NF-κB can regulate PLC-PKC signaling in platelets.

To demonstrate the physiological relevance of NF-κB in platelet activation, we investigated effects of NF-κB inhibitors on [Ca²⁺]i mobilization and platelet aggregation. BAY11-7082, at low concentration of up to 10 μM, not only significantly attenuated [Ca²⁺]i mobilization, but also inhibited platelet aggregation stimulated by collagen (Figure 4A, B). In addition, preincubation of platelets with the inhibitors, SQ22536 (100 μM) and H89 (5 μM), both strongly reversed sesamol's inhibition of [Ca²⁺]i mobilization and platelet aggregation (Figure 4A, B). Taken together, our data suggest that NF-κB is involved in [Ca²⁺]i mobilization and platelet aggregation, and sesamol's inhibition of NF-κB is mediated, at least partly, by a cyclic nucleotide-dependent pathway.