Resveratrol and platelet aggregation / thrombosis

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Platelets contribute to the rate of development of atherosclerosis and CHD through several mechanisms. It has been shown that resveratrol reduces platelet aggregation in human platelet-rich plasma in particular after induction by thrombin and adenosine-5’-diphosphate (ADP) treatment [106-108]. These in vitro results can were found again in vivo [109]. In fact, thrombin downregulates endothelial ectonucleotidase activity resulting in high level of ADP and ATP which lead to platelet and endothelial activation. Resveratrol inhibits thrombin-induced ADP and ATP secretion from platelets, decreases neutrophil function and restores the CD39/ATPDase (ATP diphosphohydrolase) in response to thrombin [110]. Furthermore, when activated by thrombin, platelets produce ROS. This free radical generation can be reduced by resveratrol in blood platelets [111, 112]. In addition to thrombin and ADP another factor, platelet-activating factor (PAF), has been reported to be also involved in atheromatosis generation. Resveratrol was able to inhibit PAF-induced platelet aggregation [113] and its pro-inflammatory effects [29]. The PAF-induced platelet aggregation is accompanied by the release of thromboxane A₂ (TxA₂), a pro-aggregant and vasoconstrictor agent. Moreover, PAF stimulates polymorphonuclear leukocytes to aggregate, to release leukotrienes and to generate superoxide. Similarly, PAF promotes aggregation of monocytes. So, reveratrol inhibiting PAF effects can reduce the effects of pro-aggregants / pro-inflammatory agents such as eicosanoids and leukotrienes.

The synthesis of eicosanoids and leukotrienes from arachidonic acid is also linked to the platelet aggregation. The synthesis of products from arachidonic acid in human platelets occurs according to several pathways. The lipoxygenase pathways lead to hydroperoxyeicosatetraenoic acids (HPETE) which are unstable and may be converted to their corresponding hydroxy fatty acids (HETE). The cyclooxygenase (COX) and the prostaglandin H synthase (PHS) pathways lead to the cyclic endoperoxides and the subsequent metabolic products such as TxA₂.

Resveratrol is able to act on lipoxygenase family. Resveratrol inhibits both 5-lipoxygenase and 15-lipoxygenase as a competitive inhibitor [38]. Resveratrol prolonged the lag phase of both enzymes, indicating a possible reduction of Fe(III) to Fe(II) at the catalytic site [114]. Pinto et al have shown that resveratrol inhibits the dioxygenase activity of lipoxygenase and is simultaneously oxidized by the peroxidase activity of lipoxygenase. The oxidized form of resveratrol is a lipoxygenase inhibitor as efficeint as the reduced form [37, 115]. This lipoxygenase inhibition by resveratrol prevents the release of pro-inflammatory substances such as 5-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE), 5,12-dihydroxy-6,8,10,14-eicosatetraenoic acid (5,12-diHETE), 12-hydroxyeicosapentaenoic acid (12 HETE), leukotriene B4 (LTB4) and its isomers (6-trans-LTB4, 12-trans-epi-LTB4) and its glutathione-conjugated derivative (LTC4) [35, 40, 116]. The 12-lipoxygenase pathway of arachidonate metabolism, which is present in leukocytes and platelets, can be reduced by resveratrol, which blocks the synthesis of hepoxilins, mediators of calcium mobilization, vascular permeability and neutrophil activation [117, 118].

Resveratrol is also a competitive inhibitor of COX and peroxidase activity of PHS [38]. As far as PHS is concerned, both COX and peroxidase activities depend on ferriprotoporphyrin IX [119, 120]. Again, the prolonged lag phase of the COX reaction was indicative of a reduction of Fe(III) to Fe(II) [120, 121]. The COX inhibition by resveratrol prevents the release of COX products such as prostaglandins and thromboxanes. For example, the polyphenol reduces the prostaglandin synthesis, decrease HHT, TxA₂ as well as TxB₂ synthesis which are proaggregant and vasoconstrictor agents [35, 118, 122, 123]. By inhibition of PLA₂, resveratrol decreases the release of arachidonate from cell lipids and thus the synthesis of metabolites by COX and lipoxygenase pathways [123]. Moreover, the polyphenol could act on the Ca²⁺ influx, subsequently reducing the activation of PLA₂ and the aggregation process. Indeed, an increase in intracellular free Ca²⁺ is an essential component of the aggregation process in platelets. Ca²⁺ must enter the cell from the external media through specific and tightly regulated Ca²⁺ channels in the plasma membrane. It appears that resveratrol is an inhibitor of store-operated Ca2+ channels and calcium influx in thrombin-stimulated human platelets [124-126]. Moreover, the blocking of calcium ion influx into cultured murine macrophages by resveratrol is one of the possible mechanisms of the pro-inflammatory IL-6 biosynthesis inhibitory action of resveratrol [51]. Nevertheless, Slater et al. [127] found that the inhibition of PKCa activity is competitive with respect to phorbol ester concentration but non competitive with respect to Ca2+ and phosphatidylserine concentrations suggesting that resveratrol compete for phorbol ester binding site to the C1 domains.

Ca²⁺ regulates various pathways and is a major second messenger implicated in signal transduction pathways regulating cell cycle, proliferation and apoptosis. Several pro-atherogenic stimuli induce ECs apoptosis through Ca²⁺-dependent pathways and contribute to the development of vascular lesions. OxLDL-mediated endothelial cells apoptosis is dependent on an increase in intracellular Ca²⁺ [128]. Thus, alterations in intracellular Ca²⁺ in ECs may cause EC dysfunction in response to oxLDL and may influence EC response to oxLDL and inflammatory cytokines, particularly TNF-a. Resveratrol blocking the Ca²⁺influx could prevent the EC apoptosis. Several matrix elements play also an important role in platelet aggregation such as collagen and fibrinogen. Resveratrol was shown to block the first step of platelet activation by inhibiting platelet adhesion to type I collagen and to decrease collagen-induced platelet aggregation [129, 130] (figure 4). Moreover, resveratrol inhibited the messenger RNA (mRNA) expression of type I collagen [131]. At cellular level, in the platelets, resveratrol can inhibit MAPK activation induced by collagen, thrombin and ADP [132] (figure 5). So, resveratrol could block receptor-mediated signaling events in platelets.

Concerning the blood platelet adhesion to fibrinogen, another initial step of platelet activation, resveratrol inhibits adhesion of both thrombin- and ADP-activated platelets to fibrinogen [133] or after activation by LPS or LPS with thrombin [130, 134]. Moreover, resveratrol could protect against atherosclerosis by promoting fibrinolysis. Indeed the polyphenol such as others compounds (catechin, epicatechin) is able to up-regulate both tissue-type plasminogen activator (t-PA) and urikinase-type PA (u-PA) gene transcription which are fibrinolytic proteins [135].

Thrombosis plays a critical role in the development, progression and clinical after-effects of atherosclerosis. The primary initiator of thrombus that mostly consisted of platelet aggregates, is a cell surface receptor for factor VII (a), the tissue factor (TF) [136]. The reduction of TF expression in vascular cells, ECs and monocytes may also contribute to the anti-aggregatory effects of resveratrol [59, 137] (figure 4). In fact, resveratrol reduced TF activity, TFmRNA by inhibition of nuclear factor kappa B (NFkB) / Rel-dependent transcription by impairing the transactivation potential of p65 [137, 138]. The diminution of c-Rel/p65 activity was dependent upon inhibition of degradation of the c-Rel/p65 inhibitory IkBa (inhibitor of kB) [138] (figure 5). The anti-thrombosis properties of resveratrol have been also showw in vivo. Indeed, resveratrol orally administrated with a high-fat diet in genetically hypercholesterolemic mice (apoE-/-/LDLR-/-) suppressed the formation of atheroma in the aortae and reduced the laser-induced thrombosis in their carotid arteries [8].