Potent antiplatelet activity of sesamol in an in vitro and in vivo model: pivotal roles of cyclic AMP and p38 mitogen-activated protein kinase.

Sesamol is a potent phenolic antioxidant which possesses antimutagenic, antihepatotoxic and antiaging properties. Platelet activation is relevant to a variety of acute thrombotic events and coronary heart diseases. There have been few studies on the effect of sesamol on platelets. Therefore, the aim of this study was to systematically examine the detailed mechanisms of sesamol in preventing platelet activation in vitro and in vivo. Sesamol (2.5-5 µM) exhibited more potent activity of inhibiting platelet aggregation stimulated by collagen than other agonists. Sesamol inhibited collagen-stimulated platelet activation accompanied by [Ca(2+)](i) mobilization, thromboxane A(2) (TxA(2)) formation, and phospholipase C (PLC)γ2, protein kinase C (PKC) and mitogen-activated protein kinase (MAPK) phosphorylation in washed platelets. Sesamol markedly increased cAMP and cGMP levels, endothelial nitric oxide synthase (eNOS) expression and NO release, as well as vasodilator-stimulated phosphoprotein (VASP) phosphorylation. SQ22536, an inhibitor of adenylate cyclase, markedly reversed the sesamol-mediated inhibitory effects on platelet aggregation and p38 MAPK phosphorylation, and sesamol-mediated stimulatory effects on VASP and eNOS phosphorylation, and NO release. Sesamol also reduced hydroxyl radical (OH(●)) formation in platelets. In an in vivo study, sesamol (5 mg/kg) significantly prolonged platelet plug formation in mice. The most important findings of this study demonstrate for the first time that sesamol possesses potent antiplatelet activity, which may involve activation of the cAMP-eNOS/NO-cGMP pathway, resulting in inhibition of the PLCγ2-PKC-p38 MAPK-TxA(2) cascade, and, finally, inhibition of platelet aggregation. Sesamol treatment may represent a novel approach to lowering the risk of or improving function in thromboembolism-related disorders.

Comparison of the relative activities of inducing platelet apoptosis stimulated by various platelet-activating agents.

Apoptosis-like events are known to occur in anuclear platelets. Although the mechanisms responsible for these events are still not completely understood, studies suggested that some platelet agonists can activate platelet apoptosis. However, the relative activities of various platelet agonists in inducing apoptosis have not yet been investigated. In the present study we explored this issue, and attempted to identify the correlation between platelet activation and apoptosis. In a platelet aggregation study, there were no significant differences respectively stimulated by arachidonic acid (AA; 100 microM), ADP (20 microM), collagen (10 microg/mL), thrombin (0.1 U/mL), U46619 (10 microM), and A23187 (5 microM). In a subsequent study, we fixed these concentrations of agonists to further compare their relative activities in inducing platelet apoptosis. Our results found that thrombin, U46619, and A23187 possess stronger activities than the other agonists in inducing platelet apoptosis (i.e., phosphatidylserine exposure, mitochondrial membrane potential depolarization, eukaryotic initiation factor (eIF)2alpha, and caspase activation). On the other hand, AA induced no apoptotic events in platelets. Based on this approach, we demonstrated for the first time that thrombin, U46619, and A23187, but not AA, possess stronger activity in inducing platelet apoptosis. In addition, we also found that platelet activation might not necessarily be associated with the occurrence of platelet apoptosis. The in vivo physiological function of the apoptotic machinery in platelets is not yet clearly understood, and needs to be further investigated in the future.

Amyloid beta peptide-activated signal pathways in human platelets.

Amyloid beta peptide (amyloid-beta), which accumulates in the cerebral microvessels in an age-dependent manner, plays a key role in the pathogenesis of cerebral amyloid angiopathy. Platelets are an important cellular element in vasculopathy of various causes. Amyloid-beta may activate or potentiate platelet aggregation. The present study explored the signaling events that underlie amyloid-beta activation of platelet aggregation. Platelet aggregometry, immunoblotting and assays to detect activated cellular events were applied to examine the signaling processes of amyloid-beta activation of platelets. Exogenous amyloid-beta (1-2 microM) potentiated platelet aggregation caused by collagen and other agonists. At higher concentrations (5-10 microM), amyloid-beta induced platelet aggregation which was accompanied by an increase in thromboxane A2 (TxA2) formation. These amyloid-beta actions on platelets were causally related to amyloid-beta activation of p38 mitogen-activated protein kinase (MAPK). Inhibitors of p38 MAPK and its upstream signaling pathways including proteinase-activated receptor 1 (PAR1), Ras, phosphoinositide 3-kinase (PI3-kinase), or Akt, but not extracellular signal-regulated kinase 2 (ERK2)/c-Jun N-terminal kinase 1 (JNK1), blocked amyloid-beta-induced platelet activation. These findings suggest that the p38 MAPK, but not ERK2 or JNK1 pathway, is specifically activated in amyloid-beta-induced platelet aggregation with the following signaling pathway: PAR1 --> Ras/Raf --> PI3-kinase --> Akt --> p38 MAPK --> cytosolic phospholipase A2 (cPLA2)--> TxA2. In conclusion, this study demonstrates amyloid-beta activation of a p38 MAPK signaling pathway in platelets leading to aggregation. Further studies are needed to define the specific role of amyloid-beta activation of platelets in the pathogenesis of vasculopathy including cerebral amyloid angiopathy.

Inhibitory mechanisms of resveratrol in platelet activation: pivotal roles of p38 MAPK and NO/cyclic GMP.

Resveratrol has been reported to have antiplatelet activity; however, the detailed mechanisms have not yet been resolved. This study aimed to systematically examine the detailed mechanisms of resveratrol in the prevention of platelet activation in vitro and in vivo. Resveratrol (0.05-0.25 micromol/l) showed stronger inhibition of platelet aggregation stimulated by collagen (1 microg/ml) than other agonists. Resveratrol (0.15 and 0.25 micromol/l) inhibited collagen-induced platelet activation accompanied by [Ca(+2)]i mobilization, thromboxane A(2) (TxA(2)) formation, phosphoinositide breakdown, and protein kinase C (PKC) activation. Resveratrol markedly increased levels of NO/cyclic guanosine monophosphate (GMP), and cyclic GMP-induced vasodilator-stimulated phosphoprotein phosphorylation. Resveratrol markedly inhibited p38 mitogen-activated protein kinase (MAPK) but not Jun N-terminal kinase or extracellular signal-regulated kinase-2 phosphorylation in washed platelets. Resveratrol-reduced hydroxyl radical (OH(-)) formation in the electron spin resonance study. In an in vivo study, resveratrol (5 mg/kg) significantly prolonged platelet plug formation of mice. In conclusion, the main findings of this study suggest that the inhibitory effects of resveratrol possibly involve (i) inhibition of the p38 MAPK-cytosolic phospholipase A(2)-arachidonic acid-TxA(2)-[Ca(+2)]i cascade and (ii) activation of NO/cyclic GMP, resulting in inhibition of phospholipase C and/or PKC activation. Resveratrol is likely to exert significant protective effects in thromboembolic-related disorders by inhibiting platelet aggregation.

Characterization of a novel and potent collagen antagonist, caffeic acid phenethyl ester, in human platelets: in vitro and in vivo studies.

OBJECTIVE: Caffeic acid phenethyl ester (CAPE), which is derived from the propolis of honeybee hives, has been demonstrated to possess multiple pharmacological activities. In the present study, CAPE (6-25 microM) specifically inhibited collagen-induced platelet aggregation and the ATP release reaction in platelet suspensions. METHODS: Platelet aggregation, flow cytometric analysis, immunoblotting, and electron spin resonance (ESR) were used to assess the anti-platelet activity of CAPE. Fluorescein sodium-induced platelet thrombi in mesenteric microvessels of mice were used for an in vivo study. RESULTS: CAPE (15-100 microM) produced a concentration-related rightward displacement of the collagen concentration-response curve, and the Schild plot gave pA(2) and pA(10) values of 4.28+/-0.07 and 3.14+/-0.73, respectively, with a slope of -0.83+/-0.16, indicating specific antagonism. CAPE (25 microM) also inhibited platelet aggregation stimulated by the glycoprotein VI agonist, convulxin, and the alpha(2)beta(1) integrin agonist, aggretin. CAPE (25 microM) also markedly interfered with FITC-collagen binding to platelet membranes. CAPE (15 and 25 microM) concentration-dependently inhibited collagen-induced platelet activation accompanied by [Ca(+2)](i) mobilization, phosphoinositide breakdown, activation of protein kinase C and mitogen-activated protein kinases (i.e., ERK2, JNK, and p38 MAPK), Akt phosphorylation, and thromboxane A(2) formation. In the ESR study, CAPE (15 and 25 microM) markedly reduced hydroxyl radical (OH) formation in collagen-activated platelets. In an in vivo study, CAPE (5 mg/kg) significantly prolonged the latency in inducing platelet plug formation in mesenteric venules of mice. CONCLUSIONS: The most important findings of this study suggest that CAPE specifically inhibits collagen-induced platelet activation. Thus, CAPE treatment may represent a novel approach to lowering the risk of or improving function in thromboembolism-related disorders.

A potent antioxidant, lycopene, affords neuroprotection against microglia activation and focal cerebral ischemia in rats.

We investigated the effects of a potent antioxidant, lycopene, on the free radical-scavenging activity as evaluated by the DPPH test and lipid peroxidation in rat brain homogenates as well as nitric oxide (NO) formation in cultured microglia stimulated by lipopolysaccharide. In addition, we also investigated the therapeutic effect of lycopene in attenuating ischemia/reperfusion brain injury induced by middle cerebral artery (MCA) occlusion in rats. Lycopene (1, 2 and 5 microM) exerted increased DPPH decolorization in the DPPH test, and increased inhibition of iron-catalyzed lipid peroxidation (TBARS formation) in rat brain homogenates in concentration-dependent manners. Furthermore, lycopene (5 and 10 microM) significantly inhibited nitrite production by about 31% and 61% in microglia stimulated by LPS, respectively. Rats which received lycopene at a dosage of 4 mg/kg, but not at 2 mg/kg, showed significant infarct size reductions compared with those which received the solvent control (20% Tween 80). In conclusion, we demonstrate a protective effect of lycopene on ischemic brain injury in vivo. Lycopene, through its antioxidative property, mediates at least a portion of free radical-scavenging activity and inhibits microglia activation, resulting in a reduction in infarct volume in ischemia/reperfusion brain injury.

Expression of matrix metalloproteinase-9 in human platelets: regulation of platelet activation in in vitro and in vivo studies.

1. The aim of this study was to identify the presence of matrix metalloproteinase-9 (MMP-9) in human platelets and systematically examine its inhibitory mechanisms of platelet activation. 2. In this study, we report on an efficient method for the quantitative analysis of pro-MMP-9 in human platelets using capillary zone electrophoresis (CZE). To elucidate subcellular localization of MMP-9 in human platelets, we investigated intraplatelet MMP-9 by immunogold labeling and visualized it using electron microscopy. In an in vivo thrombotic study, platelet thrombus formation was induced by irradiation of mesenteric venules with filtered light in mice pretreated with fluorescein sodium. 3. MMP-9-gold labeling was observed on the plasma membrane, alpha-granules, open canalicular system, and within the cytoplasma both in resting and activated platelets. Furthermore, activated MMP-9 concentration-dependently (15-90 ng ml(-1)) inhibited platelet aggregation stimulated by agonists. Activated MMP-9 (21 and 90 ng ml(-1)) inhibited phosphoinositide breakdown, intracellular Ca(2+) mobilization, and thromboxane A(2) formation in human platelets stimulated by collagen (1 microg ml(-1)). In addition, activated MMP-9 (21 and 90 ng ml(-1)) significantly increased the formation of nitric oxide/cyclic GMP. 4. Rapid phosphorylation of a platelet protein of Mr 47,000 (P47), a marker of protein kinase C activation, was triggered by phorbol-12, 13-dibutyrate (PDBu) (60 nm). This phosphorylation was markedly inhibited by activated MMP-9 (21 and 90 ng ml(-1)). Activated MMP-9 (1 microg g(-1)) significantly prolonged the latency period of inducing platelet plug formation in mesenteric venules. 5. These results indicate that the antiplatelet activity of activated MMP-9 may be involved in the following pathways. (1) Activated MMP-9 may inhibit the activation of phospholipase C, followed by inhibition of phosphoinositide breakdown, protein kinase C activation, and thromboxane A(2) formation, thereby leading to inhibition of intracellular Ca(2+) mobilization. (2) Activated MMP-9 also activated the formation of nitric oxide/cyclic GMP, resulting in inhibition of platelet aggregation. These results strongly indicate that MMP-9 is a potent inhibitor of aggregation. It may play an important role as a negative feedback regulator during platelet activation.